Ajayi tore his ACL on Sunday against the Minnesota Vikings, a source told ESPN's Adam Schefter.
Defending-champion Philadelphia (2-3) has been hit hard by injuries at the offensive skill positions.
Receivers Mike Wallace (fractured leg) and Mack Hollins (groin) were placed on injured reserve earlier in the season, and running backs Darren Sproles (hamstring) and Corey Clement (quadriceps) have been unable to stay on the field.
Ajayi, who is in a contract year, has been playing through a transverse fracture in his back suffered against the Tampa Bay Buccaneers in Week 2.
The Eagles have been struggling to put up points even with Ajayi in the lineup, scoring fewer than 24 in each of their first five games. This comes a year after they scored 24-plus points in 12 games last season, tied for the most in the NFL.
Coach Doug Pederson dialed up just four meaningful run plays in the first half of Sunday's 23-21 loss to the Minnesota Vikings. Ajayi, who finished with eight carries for 29 yards and had a costly fumble near the Vikings' goal line Sunday, stressed postgame the importance of establishing the run.
"Obviously we want to be able to run the ball early and start that rhythm early in the beginning of the game. If I remember correctly, we had maybe three carries at the end of the first quarter," he told reporters.
"With the offensive line we have on this team, running the ball like that, that doesn't make sense to me."
The coaching staff now has to figure out how to distribute Ajayi's snaps. Sproles and Clement do not appear far off from a return -- Clement was active Sunday but did not play -- and the Eagles also have Wendell Smallwood and rookie Josh Adams on the roster. It will take a collective effort, and maybe some outside help, to fill the void.