The 24-year-old Rubio was helped off the floor and taken to the locker room for X-rays after landing awkwardly with 2:28 remaining in the second quarter. The point guard had drawn a foul on a drive to the basket.
The Timberwolves said at halftime that Rubio would not return to Friday's game.
Rubio had four points, three rebounds and six assists in 13 minutes against the Magic. He was averaging 10.8 points and 11 assists coming into the night.
He played in all 82 games for the Timberwolves last season.