"You know there's this clown Marco Rubio, I've been so nice to him," Trump said at the Family Research Council's Values Voter Summit in Washington, D.C.
The jab was immediately met with sustained booing from many of those in attendance.
Trump tried to talk over the boos, which lasted several seconds: "I've been so nice to him. No. But I've been so nice."
Trump and Rubio have been anything but "nice" to each other this week, engaging in a continued back-and-forth that started Tuesday.
One of Trump's top aides kept the feud going earlier on Friday on CNN.
"If there's a debate tomorrow, Donald Trump is still front and center," Trump executive vice president Michael Cohen told CNN's Alisyn Camerota on "New Day." "He's still eight times higher in the polls than Marco Rubio. He doesn't think about Marco Rubio. Marco Rubio doesn't mean anything to him."
Trump and Rubio have been taking shots at each other this week, with the latest hit landing Thursday on Fox, where the Florida senator said Trump is "very thin-skinned, obviously. He's very sensitive to criticism."
Rubio has been gaining steadily in national polls, along with former Hewlett-Packard CEO Carly Fiorina, while Trump's lead has been slipping a bit since the second Republican debate.
Rubio's blast came in response to Trump's attack on CNN earlier in the day. "Marco Rubio, he's like a kid. He shouldn't be running in this race as far as I'm concerned," Trump said.
"I don't think Mr. Trump went after Marco Rubio, Marco Rubio went after Mr. Trump. Mr. Trump responded and then, of course, Marco Rubio responded again," Cohen said Friday. "He doesn't want to pick a fight. But if you pick a fight with him, you're gonna lose."