Barkley was injured after his right ankle bent awkwardly while being tackled by Buccaneers safety Mike Edwards late in the second quarter. He limped to the sideline and was attended to by trainers.
The All-Pro running back was helped to the locker room and ruled out for the game. He was later seen on the Giants' sideline in the second half wearing a walking boot and using crutches.
At the time of his departure, Barkley had 10 rushing yards on eight carries and 27 receiving yards on four receptions. He entered Sunday second in the NFL with 227 rushing yards, averaging 7.8 yards per carry.
The Giants, led by rookie quarterback Daniel Jones, scored 22 points in the second half to earn a 32-31 comeback victory over the Bucs.