The second-year running back went one-on-one against a Buccaneers defender and appeared to land awkwardly after he was tripped up.
Barkley was in visible pain as he was being helped off the field on Sunday.
The Giants initially announced Barkley injured his ankle on the play, and he was questionable to return before later ruling him out for the remainder of the NFL contest.
Before leaving the game, Barkley had eight carries for 10 yards.
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