At an event in Iowa on Wednesday he asked the audience "should we keep them in the room?"
The comment, made after he welcomed the 'Christian Conservatives for Trump' group, was taken as a joke in the hall.
He asked conservative Christians to raise their hands before doing the same with those who were not.
"Raise your hand, Christian conservatives, everybody. Raise your hand if you're not a Christian conservative. I want to see this, right. Oh, there's a couple people, that's all right," said Trump.
"I think we'll keep them. Should we keep them in the room? Yes? I think so."
The Council on American-Islamic Relations criticised the comments.
National communications director Ibrahim Hooper said: "Given Mr. Trump's history of targeting religious and ethnic minorities, it is disturbing that he would single out non-Christians during his rally in Iowa.
"It is clear that his vision of America is one of division and exclusion, not unity and acceptance."
