Pugh practiced Wednesday in a limited capacity but was kept out of Thursday's workout and has been listed as out for Sunday's game against the Cleveland Browns.
It will be the third straight game that Pugh has missed with a sprained MCL in his right knee.
"He got sore. He had a little setback," Giants coach Ben McAdoo said. "We'll wait to see where he is next week."
Pugh was originally expected to be sidelined two to four weeks with the injury. Next week's game against the Pittsburgh Steelers always appeared to be the realistic target for his return.
The Giants will re-evaluate Pugh early next week, with the Steelers and then the Dallas Cowboys on the horizon and first place in the NFC East potentially at stake.
In the meantime, the Giants will start their fourth left guard in four weeks, with Adam Gettis getting the call against the Browns. Pugh, Brett Jones (calf) and Marshall Newhouse (knee) already have been ruled out.