He said that Mrs. Clinton would put liberal justices on the Supreme Court if she wins the presidency in November, threatening gun ownership rights.
Speaking at a rally in North Carolina, Mr. Trump hinted that gun rights advocates could stop her taking power.
That sparked an online backlash, many accusing him of inciting violence.
He replied that he was only urging gun rights supporters to vote in large numbers.
But House Speaker Paul Ryan, the highest-ranked elected Republican, called it an inappropriate joke.
The remarks that sparked the firestorm were made at a rally in Wilmington on Tuesday afternoon.
The Republican presidential nominee said of his Democratic opponent: “Hillary wants to abolish, essentially abolish the Second Amendment. By the way and if she gets to pick her judges, nothing you can do, folks.
“But the Second Amendment people, maybe there is, I don’t know.”
The Second Amendment enshrines the right to bear arms in the US Constitution.
A man sitting behind Mr. Trump as he made the remarks assumed a look of disbelief as he heard them.
Once again, Donald Trump’s off-the-cuff style of speaking during his rallies has set the presidential campaign ablaze.
The Republican nominee said that gun rights advocates could do something about Mrs. Clinton after she gets elected and tries to appoint judges. What could that be? His campaign’s explanation that they would organise and vote simply doesn’t track.
In a political environment where Trump supporters chant “lock her up”, say Mrs. Clinton should face a firing squad or worse, the Republican candidate’s open-to-interpretation remarks likely throw gasoline onto a smouldering fire.