Spencer, a key cog in the NFL's top-ranked defense, had been listed as questionable.
The Cowboys planned for Spencer to play as late as Monday morning. He was replaced in the starting lineup by Victor Butler, a fourth-year player with eight career sacks.
Spencer, a former first-round pick playing this season under a franchise tag tender, has been a force during the Cowboys' 2-1 start. He ranks second on the team in tackles (29, according to coaches' film review) and sacks (two) and leads the Cowboys with nine quarterback hurries and two tackles for loss.
Rob Ryan's defense will be missing three other starters, including strong safety Barry Church, who was placed on injured reserve after tearing his Achilles tendon in the Week 3 win over the Tampa Bay Buccaneers.
Pro Bowl nose tackle Jay Ratliff, who had yet to play this season due to a high ankle sprain, might be able to return after the Cowboys' upcoming bye. Defensive end Kenyon Coleman will miss his second consecutive game with a hyperextended knee.