It was unclear when Spiller was hurt, but he was ineffective in the first half, gaining just 9 yards on nine carries. He rushed for no gain with 14:39 remaining in the third quarter, his last touch.
Spiller remained on the bench for most of the third quarter until the team announced his knee injury. He later departed for the locker room and was not spotted after the game.
Expectations this season were high for Spiller, who the Bills said would get a heavy workload this season. Through three games, he has rushed 43 times for 153 yards and no touchdowns.
In his place Sunday, the Bills leaned on veteran Fred Jackson, who rushed 13 times for 67 yards, 59 of which came on a third-and-1 run.
Spiller was just one of many Bills injured in Sunday's loss. Cornerback Leodis McKelvin (hamstring), as well as defensive linemen Alex Carrington (knee) and Marcell Dareus (ankle) -- all starters on defense -- left the game and did not return. Meanwhile, defensive end Mario Williams was undergoing treatment after the game for an apparent injury, but his status remains unclear.
The Bills were also without another pair of defensive starters in safety Jairus Byrd (sore feet) and cornerback Stephon Gilmore (fractured wrist), while wide receiver Marquise Goodwin (fractured hand) and cornerback Ron Brooks (fractured foot) remain on the active roster but are expected to miss "some time," according to coach Doug Marrone.