One day before the season-opener in 2003, Kolb was named the starter in Houston as a true freshman, the first true freshman quarterback to start in the season-opener in school history, making his debut against Rice at Robertson Stadium. On September 20, Kolb threw for 321 yards and four touchdowns and went 20-for-29 passing in the 42–35 win over Mississippi State. He was named Conference USA Player of the Week for his performance. On October 25, Kolb accounted for over 400 yards passing and 100 yards rushing against TCU in a 62-55 loss to the Horned Frogs, only the fourth time this feat had been achieved in Division I-A. He tied the league record with 578 total yards, and earned Conference USA offensive co-player of the week honors for his performance. Kolb finished the season with 3,131 passing yards, 25 touchdowns, and only six interceptions en route to a 7-6 record on the year, which ended with a 54-48 loss to Hawaii in the Hawaii Bowl. Following the season, Kolb was named Conference USA Freshman of the Year.

Kolb was named to the Walter Camp Award, Davey O’Brien Award and Maxwell Award watchlists prior to the start of the 2004 season. He started in all 11 games in 2004, finishing first in the conference in total offense with 256.1 yards per game, and he passed for 2,766 yards and 11 touchdowns.

Kolb was named to the Maxwell Award and Davey O’Brien Award watchlists for the second straight year in 2005. After a losing season in 2004, Kolb led the Cougars to a 6-5 regular-season record in 2005. He was named the Cougars’ MVP in the Fort Worth Bowl in a 42–13 loss to Kansas after passing for 214 yards and scoring one rushing touchdown. He finished the season with 3,258 passing yards, and became the school’s all-time total offense leader. He earned third-team All-Conference USA honors following the season.

Kolb was named to the Maxwell Award watchlist for the third consecutive year in 2006. He was named the consensus Conference USA Preseason Offensive Player of the Year, and he earned preseason all-conference honors. The 2006 season was Kolb’s signature college year, in which he threw 30 touchdowns and only four interceptions on the way to a 10–4 record and a Conference USA championship. He went 22-for-35 for 235 yards and three touchdowns in a win over Rice University on September 2, and was named the Conference USA Player of the Week for his efforts. He was named as a finalist for the Johnny Unitas Golden Arm Award on November 1. He was named the Conference USA Offensive Player of the Year on November 30. Kolb won the conference’s offensive MVP, but Houston lost to South Carolina 44-36 in the Liberty Bowl. He finished the season with 3,808 passing yards, 30 touchdowns and four interceptions. Kolb ended his career with 12,964 total passing yards, fifth all-time.

Kolb was invited to play in the 2007 East–West Shrine Game on January 20, 2007, and the 2007 Senior Bowl on January 27.

Kolb was rated as the sixth-best quarterback in the 2007 NFL Draft by NFLDraftScout.com, and was projected to be drafted in the third or fourth round. He hired Jeff Nalley and Vann McElroy as his agents prior to the draft, and they hired former NFL quarterback Jerry Rhome to work with Kolb to get him ready for the pre-draft workouts. He worked out with Danny Arnold in Stafford, Texas in February 2007 in order to prepare for the NFL Scouting Combine. Kolb worked out for the Baltimore Ravens in the first week of April, and followed it up with a visit to the Philadelphia Eagles.

Kolb was drafted by the Philadelphia Eagles in the second round (36th overall) of the 2007 NFL Draft. He was the third quarterback taken in the draft behind JaMarcus Russell (1st overall) and Brady Quinn (22nd overall). He signed a four-year, $4.285 million contract with the team on July 25, 2007. Kolb spent 2007 as the third-string quarterback behind Donovan McNabb and A. J. Feeley.

Kolb married his wife Whitney Huddleston in February 2007. Whitney gave birth to their first child, a daughter named Kamryn June, on January 10, 2009, and their second child, a daughter named Atley Rose, was born in April 2010.

Kolb’s first NFL start was a 48–22 loss against the New Orleans Saints at home on September 20, 2009 due to an injury to Donovan McNabb. His first career touchdown was recorded in the first quarter of that game on a 71-yard pass to DeSean Jackson; he threw for 391 yards on 31 completions. On September 27, 2009, Kolb made his second start in place of McNabb, throwing for 327 yards and two touchdowns in a 34–14 win against the Kansas City Chiefs. Kolb became the first quarterback in NFL history to throw for more than 300 yards in each of his first two career starts. He was named the NFC Offensive Player of the Week after his efforts against the Chiefs.

After the 2010 season, there was speculation that the Eagles would look to make a deal for Kolb since Vick was the clear starting quarterback. However, due to the 2011 NFL Labor Dispute, the Eagles were unable to trade away Kolb. On July 19, 2011, Kolb expressed interest in playing for the Arizona Cardinals, saying “Arizona would be a great place” to play.

Kolb was traded to the Arizona Cardinals on July 28, 2011, in exchange for cornerback Dominique Rodgers-Cromartie and a second-round draft pick in the 2012 NFL Draft (which the Eagles traded for draft picks used on Vinny Curry and Brandon Boykin). Kolb signed a new five-year contract worth $63.5 million with the Cardinals after the trade.

Kolb started his first game for the Cardinals on September 11, 2011, against the Carolina Panthers in the first week of the 2011 NFL season. Kolb was 18-of-27 for 309 yards, and two touchdowns in the 28–21 win over the Panthers. He went 17 for 30 passing for 251 yards, two touchdowns (including a 73-yard pass to Larry Fitzgerald) and an interception the next week in a loss to the Washington Redskins. In week 3, in a loss against the Seattle Seahawks, he had 252 yards passing on 25 completions and 39 attempts, with a touchdown and two interceptions. The next week against the New York Giants, he went 20-for-34 passing for 237 yards, one interception, and one fumble. In week 5, he had 232 yards passing and three turnovers. He went 18 for 34 with 272 yards, two touchdowns, and an interception in a fifth-straight loss during week 7 against the Pittsburgh Steelers. Against the Baltimore Ravens in week 8, Kolb suffered turf toe in the first quarter but remained in the game, passing for 153 yards on 10 completions, one touchdown, and one interception. The team lost its seventh-straight game.

In 2012, Kolb and John Skelton competed for the starting quarterback position, with Skelton being named the starter for week 1 by head coach Ken Whisenhunt. Skelton suffered an injury in the fourth quarter of the game, and Kolb led the team to a come-from-behind victory. The Cardinals began the season with four straight wins. Skelton was eventually ruled out for the Week 2 matchup against the New England Patriots. Arizona was handed their first loss of the season by the 2–2 St. Louis Rams, by a final score of 17–3. Kolb completed just around 50% of his passes, and was sacked 9 times (The 2012 Cardinals became the first team since their 2003 counterparts to allow eight or more sacks in consecutive games). In an October 14 matchup against the Buffalo Bills, Kolb suffered a rib injury late in the fourth quarter. Skelton began taking snaps for the injured Kolb and was only able to complete 2 of 10 passes with a game-costing interception in the OT period. This injury caused Kolb to miss the following seven games, all of which the Cardinals lost. Kolb had been sacked 27 times in the five starts he had. Kolb was placed on injured reserve on December 12, 2012.

The Cardinals released Kolb on March 15, 2013, after failing to agree to a restructured contract.

Kolb agreed to a two-year contract for $13 million with the Buffalo Bills on March 30, 2013. On August 24, Kolb suffered a severe concussion in a preseason game against the Washington Redskins. He was placed on injured reserve on August 30. Kolb was released on March 11, 2014.

Kolb retired in 2014 due to post-concussion syndrome, citing lingering effects from three career concussions.

On June 28, 2014, Kolb was arrested in Willacy County, Texas, for boating while intoxicated.