Apart from football, Barkley also lettered in basketball and track and field at Whitehall. As a senior in 2015, he won gold medals in the 100-meter dash (11.15 seconds) at the Eastern Pennsylvania Conference championships and the long jump (22’2.5″ or 6.77m) at the District 11 meet, where he also earned a second-place finish in the 100-meter dash with a time of 10.90 seconds which set the school’s record. Later in the season, Barkley set personal-best marks of 46’2.5″ (14.08m) in the shot put and 6’0″ (1.83m) in the high jump.

Barkley earned immediate playing time as a true freshman at Penn State in 2015. After rushing for one yard on one carry during his first game, he rushed for 115 yards with a touchdown in his second game. He followed up that performance by rushing for 195 yards and 2 touchdowns on 21 carries in his third game. In October, he missed two games due to injury. In his first game back against No. 1 Ohio State, he rushed for 194 yards on 26 carries. During his first year, Barkley had 182 carries for 1,076 yards and seven touchdowns during the 2015 Penn State football season campaign.

On November 1, 2016, Barkley was named a semi-finalist for the Maxwell Award. On November 5, Barkley rushed for 167 yards and one touchdown versus Iowa. He added 44 yards and another touchdown in the air, for a total of 211 all-purpose yards in the 41–14 win.

On December 3, 2016, Barkley had 19 carries for 83 yards, rushed for one touchdown, and caught another touchdown on a wheel route. His efforts helped Penn State recover from a 28–7 deficit to stun Wisconsin, 38–31, in the 2016 Big Ten Championship Game.

During the 2017 Rose Bowl, Barkley added 25 carries for 194 yards, at 7.8 yards per carry, as well as a 79-yard touchdown that gave Penn State a 28–27 lead. After trailing 13–0, Penn State scored seven touchdowns on seven consecutive possessions, including four touchdowns on four consecutive offensive plays, including Barkley’s 79-yard touchdown. Barkley would score the first, fourth, and seventh touchdowns for Penn State, giving the Nittany Lions a 49–35 lead in the fourth quarter, but ultimately USC won the game, 52–49.

In the 2017 Big Ten opener, the Nittany Lions visited the Iowa Hawkeyes. In the dramatic 21–19 win, Barkley had a total of 358 all-purpose yards. He also set a program record for most yards in a single game. Barkley finished the game with 211 rushing yards and a touchdown, 94 receiving yards, and 53 kick return yards. For his efforts, Barkley was again named Big Ten Offensive Player of the Week.

In the first Big Ten home game of his junior campaign against the Indiana Hoosiers, Barkley carried the ball 20 times for only 56 yards, an underwhelming rushing day for him. Despite this, he still had a major impact on the game, returning the opening kickoff 97 yards for a touchdown, adding 51 yards on 4 catches, and even throwing a 16-yard pass to DaeSean Hamilton late in the fourth quarter to cap off a 45–14 win. Barkley became the first player in Big Ten history to score a return and passing touchdown in the same game. For his efforts, Barkley was named Big Ten Special Teams Player of the Week. One month later against #6 Ohio State, Barkley again returned the opening kickoff back 97 yards for a touchdown, but was limited for the rest of the game to 44 yards rushing on 21 carries, 36 of which came on a TD run in the second quarter. In his final collegiate game, Barkley rushed for 137 yards and 2 touchdowns, one of them a 92-yard effort to put Penn State up 28–7 over the #11 Washington Huskies in the 2017 Fiesta Bowl, which Penn State would go on to win 35–28. On December 31, 2017, about 24 hours after the team’s Fiesta Bowl victory, Barkley declared his intentions to enter the NFL draft.

On December 31, 2017, Barkley, who was a projected top 10 pick by many, declared his intentions to enter the 2018 NFL Draft. In the weeks leading up to the NFL Combine Barkley’s stock rose even higher. By late February, mock drafts projected him as the second pick selection overall, the highest for a running back since Reggie Bush in 2006. Mel Kiper Jr., who has history of being against drafting running backs in the first round, said “Barkley is a once in a lifetime talent, teams that pass on this young man will be sorry.” At the combine, Barkley had a widely praised performance, running a 4.40 sec 40-yard dash and pressing 225 pounds for 29 reps, tying for the most reps at the running back position. According to Ian Rapoport, the Cleveland Browns were “strongly considering” drafting Barkley first overall, which would have made him the first running back since Ki-Jana Carter in 1995 to go first overall.

The New York Giants drafted Barkley as the second overall pick in the 2018 NFL Draft. On July 22, 2018, Barkley signed a four-year deal worth $31.2 million fully guaranteed featuring a $20.76 million signing bonus with $15 million of the bonus received immediately (remaining bonus received in October).

In 2018, the Government of Pennsylvania voted in favor of making March 14 a state holiday known as “Saquon Barkley Day”. Barkley and his family participated in a parade through his hometown of Coplay, Pennsylvania that included the Whitehall High School marching band and kids from the league where Barkley played youth football.

In Week 1 against the Pittsburgh Steelers on Monday Night Football, Barkley rushed 15 times for six rushing yards and caught six passes for 60 receiving yards during the 26–16 loss. Barkley’s 0.4 yards per attempt was a career low. During the following week’s 17–13 loss to the Chicago Bears, Barkley was carted off the field after suffering a knee injury and was later confirmed to have a torn ACL the next day, ending his season. He was placed on injured reserve on September 22, 2020.