The Catholic pontiff says Mr Trump cannot claim to be Christian while vowing to build a wall on the US-Mexico border.
It may be unprecedented - a Pope verbally crossing swords with a candidate for the United States Presidency.
On his way back from a whirwind tour of Mexico, reporters aboard Pope Francis' plane asked him about Donald Trump's plan to stop Mexicans migrating into the US by building a giant wall along the border.
"A person who thinks about building walls, wherever they may be, and not building bridges, is not Christian. As far as what you said about whether I would advise to vote, I'm not going to get involved in that. I say only that this man is not Christian if he's said things like that."
Mr Trump has been playing up his Christian values to secure a key Republican constituency - conservative evangelical Christian voters.
He's never been one to *turn the other cheek and he wasn't about to start during a speech in South Carolina.
"For a religious leader to question a person's faith is disgraceful. They're using the Pope as a pawn, and they should be ashamed of themselves. This is the Mexican government."
Some Trump supporters responded by tweeting pictures of the Vatican, surrounded by walls.
But Mr Trump's wrath didn't stop there -- he tied a debate about Christian values into the ongoing threat posed by IS militants, or ISIS.
"If and when the Vatican is attacked by ISIS, which as everyone knows is ISIS's ultimate trophy, I can promise you that the Pope would have only wished and prayed that Donald Trump would have been president."
Experts say Trump's stoush with the Pope is unlikely to harm him during this weekend's primary in South Carolina, where evangelical Protestants make up 26 per cent of the vote and Catholics only nine per cent.
But Susan Page, an analyst with news service USA Today, says it could come back to haunt him further down the track.
"I think it could be harmful to him in a general election, against Hillary Clinton or a Democrat who's making inroads among Hispanic voters or Catholics. Catholics are one of the most crucial swing votes, but when you talk about the Republican nomination I'm not sure this hurts him."
Mr Trump's Republican rivals also hold hardline policies on immigration.
Senator Ted Cruz, who's also in favour of a border wall, chose to steer clear of the issue.
"That's between Donald and the Pope, I'm not going to get in the middle of that, I'll leave it to the two of them to work it out."
Former Florida governor Jeb Bush did comment, saying questioning a person's faith is inappropriate.
"I don't question people's Christianity, that's a relationship they have with their lord and saviour and themselves, so I don't think it's appropriate to question Donald Trump's faith. He knows what his faith is. If he has a relationship with the lord, fantastic. If he doesn't it's none of my business."
The Obama administration is also sidestepping the argument, although White House press secretary Josh Earnest couldn't resist one jibe.
"I will however extend to Mr Trump the courtesy that he has not extended to the President and not use this opportunity to call into question the kind of private, personal conversations that he is having with his God."
