Kimmel used the opening monologue of Jimmy Kimmel Live! to address comments made on April 23 in a parody segment on the White House correspondents' dinner.
He said his quip that Melania Trump had "a glow like an expectant widow", referred to the difference is ages of the first lady and her husband, President Donald Trump.
"It was a very light roast joke about the fact that he's almost 80 and she's younger than I am," Kimmel said on Monday.
"It was not by any stretch of the definition a call to assassination."
Earlier in the day, Trump said Kimmel should be fired by ABC and parent company Walt Disney, joining his wife in calling out Kimmel for remarks made before a shooting near a weekend gathering of journalists and politicians.
The Trumps were rushed out of Saturday's dinner after the shooting in the lobby of the Washington Hilton.
Trump has repeatedly urged broadcasters to drop comedy or news programs he dislikes or which have been critical of him, pressing regulators to revoke licenses of broadcasters he says are unfair to him.
Broadcasters have broad constitutional rights to make jokes, however, even those that are distasteful, experts say.
Melania Trump called Kimmel's remarks "corrosive" and a symptom of what she described as a political sickness in the United States.
"I agree that hateful and violent rhetoric is something we should reject," Kimmel said.
"I do, and I think a great place to start to dial that back would be to have a conversation with your husband about it."
The comedian played a clip of a CBS News' 60 Minutes Sunday interview in which Trump called senior correspondent Norah O'Donnell "a disgrace" for reading an excerpt from the alleged gunman's writings and seeking a response.
"Enough is enough. It is time for ABC to take a stand. How many times will ABC's leadership enable Kimmel's atrocious behavior at the expense of our community," Melania Trump said in a post on X.
Donald Trump, who previously called for Kimmel to be removed from the air, said the comedian's joke was "something far beyond the pale. Jimmy Kimmel should be immediately fired by Disney and ABC."
Kimmel expressed sympathy for Melania Trump, and those who attended Saturday's event, for enduring the traumatic incident.
But he rejected the notion that a joke, delivered three days earlier, "had any effect on anything that happened".
In September 2025, the head of the Federal Communications Commission pressured broadcasters to take Kimmel off the air.
ABC briefly suspended Kimmel's show that month over comments he made about the assassination of conservative activist Charlie Kirk.
Trump, the subject of two prior assassination attempts, has repeatedly attacked the media and threatened broadcast licences.
