Charles followed up his stellar junior season by rushing for 2,056 yards and 25 touchdowns during his senior year with the Titans. The Associated Press named him to their first team all-state squad and he was declared the Houston Chronicle area offensive MVP. Charles was also named to the 2005 Parade All-America Football Team and was the District 22-5A Player of the Year both his junior and senior year. Charles participated in the 2005 U.S. Army All-American Bowl.

On March 11, 2006, Charles placed fourth in the NCAA 60-meter indoor track and field championship finals. On May 14, 2006, Charles captured his first conference title and the third Big 12 100-meter title for Texas by winning the event in 10.23 at the Big 12 outdoor meet. He led the 200 meter race after the preliminary round but elected not to participate in the final, as Texas had the Big 12 team title well in hand. On June 10, at the NCAA outdoor competition, Charles took fifth place in the 100 meter finals, edging out UTEP’s stand-out sprinter Churandy Martina (sixth place), who earlier in the year ran a 9.76 (wind-aided) 100-meters. Charles also placed seventh in the 200 meter finals, and ran the third leg of the 4 × 100 Texas Longhorn relay team, earning a fifth place in the finals. Charles’s efforts helped the Longhorns earn a third place showing for the men’s track and field team, the highest since a second-place finish at the 1997 NCAA finals. Thus, Charles completed his first collegiate track season as a four-time All-American (60m indoor, 100m outdoor, 200m outdoor, 4 × 100 m relay outdoor).

In the 2006 season, the Longhorns’ rushing offense dropped significantly from the prior year going from 274.9 yards per game to 162.6 yards per game. Despite the down year for the rushing offense and sharing the backfield with Selvin Young, Charles’s production remained consistent compared to his freshman year. He went over 100 rushing yards once, which was against in-state rival Rice in the Longhorns’ third game, a 52–7 victory. He found the endzone on the ground twice on November 11 against Kansas State in a 45–42 loss. He had a 72-yard touchdown reception from quarterback Colt McCoy in the 2006 Alamo Bowl 26–24 victory over Iowa. Overall, he finished with 831 rushing yards, seven rushing touchdowns, 18 receptions, 183 receiving yards, and one receiving touchdown.

In 2007, Charles rushed for 1,619 yards, with an average of 6.3 yards per carry. Charles started the season with 27 carries for 112 rushing yards and a touchdown in a 21–13 victory over Arkansas State. In the next game, a 34–13 victory over rival TCU, he had 22 carries for 134 rushing yards and a rushing touchdown. In the next game, a 35–32 victory away victory at Central Florida, he had 22 carries for 153 rushing yards and a rushing touchdown. In the next game, he had 14 carries for 72 rushing yards and three rushing touchdowns in a 58–14 victory over Rice. Despite his successes early in the season, Mack Brown and Greg Davis hinted that Charles could face less playing time as a result of his fumbling problems. Charles said that he felt a deep remorse over his fumbles and felt that he is the biggest reason the team lost to the Oklahoma Sooners in the 2007 Red River Shootout. Texas running backs coach Ken Rucker and former Longhorn running back Earl Campbell both worked with Charles on his ball handling. Greg Davis said he wanted to get the ball to Charles “in space”, on pitches and passes, instead of in heavy traffic up the center.

On October 28, 2007, Charles rushed 33 times for 290 yards in the 28–25 victory, the most ever against the Nebraska Cornhuskers and the fourth-highest total in Texas Longhorns history. His 216 yards in the fourth quarter were just six shy of the NCAA record for a single quarter set by the University of Washington Huskies’s Corey Dillon in 1996. For these accomplishments, Charles won a fan vote for AT&T All-America Player of the Week. He followed that up with 16 carries for 180 rushing yards and three rushing touchdowns in a 38–35 victory over Oklahoma State. On November 10, against rival Texas Tech, he had 23 carries for 174 rushing yards and a rushing touchdown in the 59–43 victory. In the Holiday Bowl against Arizona State, he had 27 carries for 161 rushing yards and two rushing touchdowns in the 52–34 victory.

Charles decided to forgo his senior season with Texas in favor of joining the NFL as a professional football player in the 2008 NFL Draft. This decision came after earlier statements that he would stay with Texas. In November, Charles said he would return for his senior season rather than enter professional football in the NFL. Sources reported in December 2007 that Charles and fellow Longhorn Jermichael Finley had filed paperwork with the NFL to evaluate how high they might be drafted if they decided enter professional football in the NFL Draft instead of returning for their senior season. Mack Brown did not comment about specific players but said, “We always try to help our guys get as much information as possible when it comes to the NFL. We encourage and help them go through the process… All of our underclassmen have told us they will be coming back, but if you’re playing well enough to be considered an NFL prospect, going through the process can only help you better understand it and realize what you need to work on to improve your status.” Charles said he would not go pro unless he was predicted to be chosen in the first round of the draft. After the Holiday Bowl, Charles said, “Right now, I’m probably coming back. I didn’t think I did that good in the game. Next year maybe I’ll be up for the Heisman. I will come back.” From 2000–2007, the Longhorns have had seven players taken in the Top 10 draft picks by the NFL, more than any other school. On January 2, Charles announced he received and was happy with the results of his draft evaluation and that he would declare for the draft.

In the 2010 season, Charles shared the majority of backfield carries with Thomas Jones. The Chiefs opened up the 2010 season with a 21–14 win over their division rival, the San Diego Chargers. In that game, Charles played a pivotal role, including a 56-yard rushing score. On October 31, against the Buffalo Bills, he had 22 carries for 177 rushing yards in the 13–10 victory. On November 28, against the Seattle Seahawks, he had 22 carries for 173 rushing yards and a rushing touchdown in the 42–24 victory. In the following game, he had 116 rushing yards in the 10–6 victory over the Denver Broncos. in Week 15, against the St. Louis Rams, he had 11 carries for 126 rushing yards and a rushing touchdown in the 27–13 victory. Charles finished the season with 1,467 rushing yards and five rushing touchdowns on only 230 carries. In addition, he had 45 receptions for 468 receiving yards and three receiving touchdowns. He was voted to his first career Pro Bowl. His 6.38 yards per carry average for the season was the second-highest average in NFL history, second only to Hall of Famer Jim Brown, only two one-hundredths of a yard off of the record pace set by the Browns legend.

On December 11, 2010, the Chiefs reached an agreement with Charles on a five-year, $32.5 million deal that included $13 million guaranteed. Charles made his postseason debut in the Wild Card Round against the Baltimore Ravens. He had nine carries for 82 rushing yards and a rushing touchdown in the 30–7 loss. In the 2011 Pro Bowl, Charles rushed for 72-yards on 10 carries and scored one touchdown. Charles was honored for his performance in the 2010 season by being selected to his first AP All-Pro team. On the NFL Network’s Top 100 Players of 2011, Charles was ranked the 33rd by his fellow players and was also one of the youngest players on the list.

Charles’s 2011 season was short lived. In a Week 1 loss to the Buffalo Bills, Charles rushed for 56 yards on 10 carries and had a receiving touchdown. The following week, against the Detroit Lions, Charles ran for 27 yards on two carries, before suffering an ACL injury. Chiefs head coach Todd Haley confirmed the next day that Charles would miss the remainder of the 2011 season. Charles was officially placed on injured reserve on September 19.

Coming off of an ACL injury in 2011, Charles had an All-Pro caliber season, running for 1,509 yards and five touchdowns. Once again, Charles proved to be very efficient running the ball as he managed a 5.3 yard per carry average on the year. In the season opening 40–24 loss to the Atlanta Falcons, he had 87 rushing yards on 16 carries in his first action back from injury. In Week 3 against the New Orleans Saints, Charles ran a career-high 33 times for 233 rushing yards, scored a rushing touchdown, and caught six passes for 55 yards to earn his first AFC Offensive Player of the Week nod. Charles became the first player in franchise history to rush for at least 200 rushing yards and have at least 50 receiving yards in the same game. His 91-yard rushing touchdown in the third quarter was the longest rushing play in franchise history, a feat later tied by Damien Williams in the 2019 season. In Week 5 against Baltimore Ravens, Charles had 31 carries for 140 rushing yards in the 9–6 loss. However, in Week 8, Charles carried the ball only five times for four rushing yards in a 26–16 loss to the Oakland Raiders. When asked the reason why, head coach Romeo Crennel said “Now, that I’m not exactly sure, either.” In Week 10, against the Pittsburgh Steelers, he had 23 carries for 100 rushing yards in the 16–13 loss. On November 25, in Week 12 against the Denver Broncos, he had 23 carries for 107 yards in the 17–9 loss. In a 30–7 loss to the Cleveland Browns later in the season, Charles carried the ball 18 times for 165 rushing yards and a rushing touchdown. On December 2, against the Carolina Panthers, he had 27 carries for 127 rushing yards in the 27–21 victory. In a 20–13 loss to the Indianapolis Colts, Charles carried the ball 22 times for 226 rushing yards and a rushing touchdown. He became the second player in franchise history, joining Larry Johnson, to record two games with at least 200 rushing yards in a single season. Charles’s performance was his second career game with over 200 rushing yards while averaging more than ten yards a carry. He became only the second player in NFL history to accomplish the feat, after Adrian Peterson, who accomplished his feat in the 2012 season as well. Charles carried the ball at least 20 times in six games, and in each of those games managed at least 100 yards. This has been a trend throughout his entire career, in every game he has carried the ball 20 times, he has gained at least 100 yards. On December 23, 2012, following his qualifying 750th career carry, Charles broke NFL legend Jim Brown’s 47-year-old all-time average yards per carry record of 5.22 with an average of 5.82. He was named to his second Pro Bowl as a result of his successful season. On the NFL Top 100 Players of 2013, he was ranked 20th by his peers.

On July 23, 2014, one day after threatening to hold out of training camp, Charles agreed to a two-year, $18.1 million extension making him one of the top five highest paid running backs in the NFL. On September 14, 2014, Charles suffered a high ankle sprain in Week 2 against the Denver Broncos. On September 29, against the New England Patriots, he had 18 carries for 92 rushing yards and a rushing touchdown in the 41–14 victory to earn AFC Offensive Player of the Week honors. On October 19, against the San Diego Chargers, Charles passed Priest Holmes’s mark of 6,070 rushing yards to become the all-time leading rusher for the Chiefs. In the next game, he had 67 rushing yards and two rushing touchdowns in the 34–7 victory over the St. Louis Rams. On November 16, against the Seattle Seahawks, he had 159 rushing yards and two rushing touchdowns in the 24–20 victory. On December 7, against the Arizona Cardinals, he had 91 rushing yards, one rushing touchdown, two receptions, 20 receiving yards, and a receiving touchdown in the 17–14 loss. Overall, he finished the 2014 season with 1,033 rushing yards, nine rushing touchdowns, 40 receptions, 291 receiving yards, and five receiving touchdowns. He tied for the team lead in receiving touchdowns, with Travis Kelce, for the second time in his career. He was named to his third consecutive Pro Bowl for the 2014 season. He was ranked 12th by his fellow players on the NFL Top 100 Players of 2015.

Charles has reported being diagnosed with a learning disability as a child, having difficulty reading, and being mocked and teased. In 2015, he shared his experience including being invited to participate in the Special Olympics: “[…] I was afraid. I was lost. When I was a boy, I had trouble reading. I found out I had a learning disability. People made fun of me. They said I would never go anywhere. But I learned I can fly. When I was 10 years old, I had a chance to compete in the Special Olympics. That’s right, the Special Olympics gave me my first chance to discover the talent I did not know that I had. When I competed in the Special Olympics, I found out just how fast I was. I stood high on the podium, getting the gold medal in track and field.”

Heading into the 2016 season, Charles never fully recovered from his torn ACL suffered the previous season. He returned to the field in Week 5 and played in three games recording 40 rushing yards plus a rushing touchdown along with two receptions for 14 yards. He suffered a setback with his knee prior to Week 8 and needed a second surgery on his knee to trim his meniscus. He was placed on injured reserve on November 1, 2016.

On February 28, 2017, Charles was released by the Chiefs.

On May 2, 2017, Charles signed a one-year, $3.75 million contract with the Denver Broncos. He was part of a backfield rotation that was shared with C. J. Anderson and Devontae Booker. In the season opener against the Los Angeles Chargers on Monday Night Football, Charles had 10 rushes for 40 yards but lost a fumble in the 24–21 victory.. In Week 3, in a 26–16 loss to the Buffalo Bills, he had nine carries for 56 rushing yards and scored his lone rushing touchdown of the season. In 14 games, he had 296 rushing yards, one rushing touchdown, 23 receptions, and 129 receiving yards.

Charles signed with the Jacksonville Jaguars on October 9, 2018. He was released on October 22, 2018. He played in two games with the Jaguars in the 2018 season and only totaled six carries for seven rushing yards and two receptions for seven receiving yards.

On May 1, 2019, Charles retired after signing a one-day ceremonial contract with the Chiefs. After signing the ceremonial contract, he went to the practice field for a ceremonial “last carry”, which he took from Patrick Mahomes.