Carles Puyol, the 34-year-old Spanish international limped off the Nou Camp pitch during the second half of Barcelona's 4-1 win over Getafe in La Liga on Saturday.
Puyol underwent knee surgery in May following an injury in the Catalan derby against Espanyol, an operation that kept the Spain international out of their victorious Euro 2012 campaign.
Making only his second appearance following his recovery, Puyol appeared to twist his knee sharply in the 52nd minute of play, although he managed to return to action shortly after.
It proved too much for the stopper to take, however, and was subsequently replaced in the 57th minute by defensive colleague Javier Mascherano.
A protracted injury for Puyol could enforce a shortage of centre-backs on Barcelona boss Tito Vilanova, who only took charge of the La Liga runners-up this summer.