The injury is a high-ankle sprain, a source told ESPN's Adam Schefter. Barkley will reportedly undergo an MRI on Monday.
He needed assistance to get to the locker room during Sunday's win over the Tampa Bay Buccaneers, and Barkley appeared to be limping heavily.
The 22-year-old exited a few minutes before halftime, and the Giants quickly announced he wouldn't return.
Barkley came back to the sideline on crutches and with a boot on his ankle:
A Buccaneers defender who was attempting to make a tackle twisted the dynamic back's ankle. Barkley tried to walk off the field, but he was unable to get to the sideline before going down again.
The second-year pro was struggling before his exit, recording eight carries for 10 yards and four catches for 27 yards, and he also dropped a would-be touchdown reception.
With Barkley out, the Giants leaned on backup Wayne Gallman as rookie quarterback Daniel Jones made his regular-season debut.