Barkley was injured during the Giants' 17-13 defeat to the Chicago Bears at Soldier Field on Sunday after falling to the turf in agony during the second quarter.
The Giants said in a statement on Monday that an MRI scan had revealed a torn anterior cruciate ligament in Barkley's right knee.
"Barkley will undergo surgery to repair the injury in the near future," the Giants said.
While the recovery time for ACL tears varies, Barkley is all but guaranteed to be out for the rest of the NFL season.
Players with similar injuries in recent years have faced a minimum of six months on the sidelines.
Barkley's absence deprives the Giants of one of the team's principal offensive weapons.
The 23-year-old running back has topped 1,000 rushing yards in each of his first two full seasons in the NFL after being drafted by the Giants in 2018 with the second overall pick.