Donald Trump called for Hillary Clinton's Secret Service agents to be stripped of their firearms - and then said: "Let's see what happens to her."
The Republican presidential candidate has long incorrectly suggested his Democratic opponent wants to overturn the Second Amendment and take away Americans' right to own guns.
At a rally in Miami, he told the crowd: "I think that her bodyguards should drop all weapons. They should disarm, right?
"Take their guns away, she doesn't want guns. Take their - and let's see what happens to her. Take their guns away. OK, it would be very dangerous."
Mr Trump later disputed that criticism, saying everyone in his audience knew he was referring to the power of voters and "there can be no other interpretation".
The tycoon, who has the endorsement of the National Rifle Association, eventually used Twitter to say the Secret Service had not contacted him about the remarks.
Crooked Hillary wants to take your 2nd Amendment rights away. Will guns be taken from her heavily armed Secret Service detail? Maybe not!— Donald J. Trump (@realDonaldTrump) September 17, 2016
The Clinton campaign reacted quickly, with spokesman Robby Mook saying Mr Trump "has a pattern of inciting people to violence. Whether this is done to provoke protesters at a rally or casually or even as a joke, it is an unacceptable quality in anyone seeking the job of Commander in Chief.
"This kind of talk should be out of bounds for a presidential candidate."
A spokeswoman for the Secret Service declined to comment.
The seemingly ominous comment echoed a remark Mr Trump made last month that many Democrats condemned as a call for Mr Clinton's assassination.
Speaking at a rally in North Carolina, the tycoon erroneously said his opponent wants to "abolish, essentially, the Second Amendment".
He continued: "By the way, if she gets to pick her judges, nothing you can do, folks.
"Although the Second Amendment people, maybe there is, I don't know."
Within minutes, the Clinton campaign condemned the remark. Mr Mook said then: "A person seeking to be the president of the United States should not suggest violence in any way."
Mr Trump's comments on Friday in Miami came hours after Mr Trump finally reversed his long-held position that President Barack Obama was not born in the United States.
Donald Trump: "President Barack Obama was born in the United States. Period." https://t.co/1e54EY3AHL— NBC News (@NBCNews) September 16, 2016
Appearing in Washington, he said Mr Obama was born in the US but then incorrectly suggested that the Clinton campaign had started the conspiracy theory.
Mr Trump ignored questions from reporters about his switch and has yet to explain why he abandoned the "birther" stance that fuelled his political fame and was viewed by critics as an attempt to delegitimise the nation's first African-American president.
WATCH: Trump ignores press as they ask questions about Trump's sudden flip-flop on Obama birth conspiracy theories. https://t.co/M4uNevu7Rs— NBC Nightly News with Lester Holt (@NBCNightlyNews) September 16, 2016
While campaigning in South Florida, which has a large Cuban-American population, Mr Trump said if he is elected president, he will reverse Mr Obama's efforts to normalise relations with Cuba - unless the country abides by certain "demands".
Among those, he said, would be religious and political freedom for the Cuban people and the freeing of all political prisoners.
Mr Trump says he will "stand with the Cuban people in their fight against communist oppression".
The comment marks yet another U-turn for the Republican candidate, who previously said he supported the idea of normal relations, but wished the US had negotiated a better deal.