Barkley was carried off the field by trainers after a run early in the first quarter. He left the game against the Chicago Bears and appeared to be in serious pain.
The Giants said Barkley underwent X-rays on his knee. He will have more tests Monday to determine the extent of the injury. One source said a torn ACL was "certainly within the realm."
Coach Joe Judge said there was no official diagnosis after the Giants lost 17-13 to the Chicago Bears at Soldier Field.
"Obviously we're all praying for the best," Judge said. "I would just say this: Regardless of whatever the outcome is going to be and the doctors say [Monday], don't fall asleep on [Barkley]. It's going to be a hell of a story either way."
Barkley was hurt as he ran toward the sideline and was being tackled by Bears safety Eddie Jackson. He grabbed at the knee as he was headed to the ground.
Barkley was later carted to the locker room and slammed his fist in disgust. He had four rushes for 28 yards before exiting. His season will likely end with 19 rushes for 34 yards and no touchdowns.
The injury came moments after Barkley was forced to miss a play when he landed on his left arm. He sat out the final play of the first quarter as he was examined on the sideline.
The Bears had their own running back issues. Running back David Montgomery tried to hurdle a lineman, was clipped and tackled with his head hitting the turf first. He went to the medical tent and then to the locker room.
The Bears said he had a neck injury, but he returned in the second half and received a bunch of carries down the stretch. Montgomery finished with 82 yards on 16 carries and a touchdown reception.
Shepard tried to return in the third quarter, but could not finish his route on the first play of the second half. He finished with two catches for 29 yards.
So much for quarterback Daniel Jones having all his top weapons healthy. Sunday was the first game that Jones had Barkley, Shepard, tight end Evan Engram and wide receivers Darius Slayton and Golden Tate all at his disposal. But it lasted only a little more than a quarter with Barkley and Shepard being injured before halftime.
"Certainly a tough break for us," Jones said after completing 25 of 40 passes for 241 yards with an interception and a lost fumble. "But I know those guys will battle. They'll attack the challenge, and we're excited to support them and we'll have guys step up."
It has been a rough start to the season for Barkley and the Giants. Barkley rushed for 6 yards on 15 carries in the opener and was criticized publicly by former Giants great Tiki Barber for his blocking. Barkley was hit behind the line of scrimmage on 11 of the 15 carries, and had eight negative runs in the opener.
Barkley's first run again went for negative yards in Chicago, and the Giants trailed 10-0 when he left. They trailed 17-0 at halftime before rallying to give themselves a chance to win the game on the Bears' 10-yard line with four seconds remaining. Jones' last-second pass to Golden Tate was incomplete and a flag was thrown on the play for offensive pass interference.
Barkley, 23, is in his third professional season after being selected with the second overall pick in the 2018 NFL draft. After a huge rookie season in which he accumulated more than 2,000 total yards from scrimmage, he was hampered by a high ankle sprain last year. He missed three games and was not at 100 percent in several others.
The Giants were expected to build their offense this season under Judge and new offensive coordinator Jason Garrett around Barkley, the talented back whom general manager Dave Gettleman considered a transcendent talent and future "gold jacket" player when he was drafted. He seemed destined to have a massive year as the centerpiece of the Giants' plans.
Now, they will have to recalibrate their offense without their top playmaker and hope he returns to his previous form by the start of next season. The extent of the further testing and subsequent damage will be the determining factors.
"I mean, he's Saquon Barkley. You lose a guy like that, it's a huge loss," said running back Dion Lewis, who took Barkley's spot in the lineup and rushed for a touchdown. "As a team, everybody has to step up. Receivers got to step up. O-line has to step up. The running backs have to step up. The quarterback has to step up. Defense has to step up. It's not going to be one person step up to make up for what he's done. It's going to take all of us."
Information from ESPN's Adam Schefter was used in this report.