Shepard was injured in last week's season-opening loss to the Dallas Cowboys. He led all Giants receivers by playing 99% of the offensive snaps.
"He just couldn't quite make it back," coach Pat Shurmur said, noting that Shepard made progress but is still in the concussion protocol. "He's getting better. So just ran out of time this week."
The Giants are also without Golden Tate, who is in the midst of serving a four-game suspension for violating the league's policy on performance-enhancing substances. Cody Latimer had been ruled questionable to play due to a calf injury, but is active.
It likely means an even bigger role in the passing game for tight end Evan Engram and running back Saquon Barkley. Engram led all Giants receivers with 11 catches on 14 targets for 111 yards and a touchdown against the Cowboys. Barkley had only 15 total touches, the second fewest of his career.
The Giants (0-1) are without two of their top receivers against a Bills defense that was tops in the NFL last season against the pass. It's a lot of potential production to be missing.
"That goes without saying," Shurmur said. "You play with the players you have, and anybody you put out there you expect them to make plays."
Veterans Bennie Fowler, Russell Shepard and T.J. Jones will likely be asked to play significant roles come Sunday. Fowler had five receptions for 40 yards in the opener, Russell Shepard wasn't targeted, and Jones was re-signed this week after getting cut several weeks back. He spent most of the summer with the Giants and is familiar with their offense.
Sterling Shepard served as the Giants' No. 1 receiver and led all wide receivers with six catches against the Cowboys. Latimer led the team with 74 receiving yards, which included a 43-yard reception.
Latimer injured his calf Wednesday in practice. He was able to run some routes on Friday, although not at full speed.
"I think I'm good to go," Latimer said. "We'll see before the game when I start running around, move around and make a decision then."
Sterling Shepard is the biggest blow. He was expected to be the Giants' top receiver with Odell Beckham Jr. traded to Cleveland this offseason and Tate suspended. Shepard had a career-best 66 catches for 872 yards last year and was signed to a lucrative long-term contract extension this offseason.
The injuries could force the Giants to alter their approach this week. They ran three-wide-receiver sets on 76% of their offensive plays on Sunday. They ran only 9.1% of their plays with two or more tight ends.