Isaac drove the lane and did a hop step between two defenders against the Sacramento Kings. He then fell to the floor in pain, untouched, while clutching his left knee with 9:19 left in the fourth quarter of the Magic's 132-116 win.
He had his head in his hands as he was wheeled off the court accompanied by Magic president of basketball operations Jeff Weltman.
It was Isaac's second game back since he hyperextended his left knee Jan. 1 at Washington. Isaac, who missed 31 games because of the injury, left Sunday's game with 4 points, 4 assists, 3 rebounds and 2 steals in 15 minutes.
Weltman told reporters on Monday that he believes "absolutely, unequivocally" that the ACL tear is not related to Isaac's previous injury and remained adamant that he was not brought back too early.
"Those of you that follow us locally know if you're ever going to criticize us with the timing of returning our players to play, it's at the other end of the spectrum," Weltman said. "It's that we're too cautious, we wait too long, we take too careful of an approach. The approach to Jonathan's injury was no different than any other injury we're had in the last three years. We were very careful."
Even though the Magic routed the Kings in Sunday's game, the Orlando bench was subdued following Isaac's injury.
"That was tough, man," teammate Aaron Gordon said. "That one brought me to tears, and instantly -- just because I know how good of a guy J.I. is and how hard he works and how hard he has worked to get back since hurting himself in D.C. That was tough."
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."
ESPN's Tim Bontemps, Nick Friedell and Royce Young contributed to this report.