Rafinha played on until the end of Barca’s 1-1 draw with Atletico Madrid on Saturday, despite appearing to feel discomfort shortly after coming off the bench in the 46th minute.
Sergi Roberto had been the player replaced, with Barcelona also confirming on Sunday he has a hamstring injury and is expected to be out for three to four weeks.
Barcelona did not offer a timescale for Rafinha’s recovery but anterior cruciate ligament injuries often take several months to heal.
A club statement said: “Rafinha has a rupture of the anterior cruciate ligament of the left knee. In the next few days we will proceed with surgical treatment.”
Sergi is likely to miss Barca’s remaining Champions League group games against PSV Eindhoven and Tottenham, as well as La Liga fixtures at home to Villarreal and away to Espanyol.
On Sergi, the club added: “Sergi Roberto will be out for between three to four weeks with a rupture of the hamstring in his left leg.”
Unlike Sergi, Rafinha has not been a regular in Barcelona’s line-up this season, but the Brazilian enjoyed a run in the side while Lionel Messi was injured last month.
He started against both Real Madrid and Rayo Vallecano in the league, as well as at home against Inter Milan in the Champions League, scoring the opening goal in a 2-0 win.
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