Isaac drove the lane and did a hop step between two defenders. Then he immediately fell to the floor in pain, untouched, while clutching his left knee with 9:19 left in the fourth quarter.
Orlando has fears Isaac tore his ACL, a source told The Undefeated's Marc J. Spears.
Isaac had his head in his hands as he was wheeled off the court accompanied by Magic president of basketball operations Jeff Weltman.
This was Isaac's second game back since he hyperextended his left knee on Jan. 1 at Washington. Isaac, who missed 31 games because of the injury, left the game with 4 points, 4 rebounds and 2 steals in 15 minutes.
Orlando head coach Steve Clifford said the team is awaiting an update on Isaac's injury.
"I'm hopeful," Clifford said after the game. "It didn't look good. It looked bad, obviously. Until you hear anything, I am just hopeful. That's all."
Clifford said Isaac will not have to leave the NBA campus to have his injury examined.
Even though the Magic routed the Kings, the Magic bench looked subdued following Isaac's injury. Teammate Aaron Gordon said the injury brought him to tears.
"That was tough, man," Gordon said. "That one brought me to tears and instantly -- just because I know how good of a guy JI is and how hard he works and how hard he has worked to get back since hurting himself in D.C. That was tough.
"I hope it's just a tweak. I hope it is nothing serious. ... I hope it is not as bad as it seems."
Kings big man Harry Giles III, who tore both of his ACLs in high school and had a procedure on his left knee before starting his college career at Duke, said he spoke to Isaac briefly before he left the floor.
"Just kind of telling him to stay up, stay positive," Giles said. "I just hate to see that because it's tough. I don't really like speaking on that too much. It's just unfortunate, man, because he's been working and with the brace on. You hate to see him get hurt again."
"For him to go down like that was very difficult to see, especially when you know he just came back," Vucevic said. "Hopefully it is nothing too serious and he doesn't have to be out for a long time because it would really suck for him, and of course we need him as well."
ESPN's Tim Bontemps and Nick Friedell contributed to this report.