Mixon attended and played college football for the University of Oklahoma from 2014–2016. He was suspended from the Oklahoma football team for the 2014 season after a misdemeanor assault charge in October 2014. He earned playing time his redshirt freshman year, playing in all 12 games with three starts. In his first career game, Mixon had a 76-yard touchdown reception, the longest catch ever by a Sooner freshman running back. In addition, he ran for 27 yards on five carries against Akron. On October 24, against Texas Tech, he had 154 rushing yards and two rushing touchdowns. In the annual rivalry game against Oklahoma State, he had 136 rushing yards and two rushing touchdowns. Oklahoma qualified for the College Football Playoff in the 2015 season. In the semifinals against Clemson in the Orange Bowl, he was limited to four rushing yards and 11 receiving yards in the loss. During the 2015 regular season, he ran for 749 yards on 110 carries, averaging 6. 8 yards. He rushed for seven touchdowns and set his career long carry at 66 yards. He also recorded 25 receptions for 345 yards, averaging 13. 8 yards per reception, and four touchdowns.