Leafs coach Randy Carlyle says Bernier is out three weeks with a medial collateral ligament sprain.
Bernier left in the third period of Toronto's 4-3 overtime win over Boston Thursday night after falling awkwardly on his left leg.
James Reimer made 10 saves on 11 shots in relief.
Bernier was making his fifth straight start since returning from a lower body injury that kept him out of five games.
The injury means Reimer will be counted on to keep the Leafs' slim playoff chances alive. Toronto trails Columbus by one point for the final playoff spot in the Eastern Conference, but the Blue Jackets have played two fewer games.
The Leafs are expected to recall Drew MacIntyre from the AHL's Toronto Marlies to fill the second goaltender spot on the bench.