Donald Trump's lawyer says Sadiq Khan should be "ashamed of himself" for criticising the US president.
Rudy Giuliani, the former New York mayor, advised the Mayor of London to focus on leading the capital rather than making comments about Mr Trump.
He said: "The Mayor of London is rallying against the president of the United States? He should be ashamed of himself. Maybe he should do his job rather than attack a world leader."
Mr Trump and Mr Khan have clashed repeatedly since the pair were elected around two years ago.
Their first public sabre rattling was after the London Bridge terror attack last summer.
Mr Trump tweeted at the time: "At least 7 dead and 48 wounded in terror attack and Mayor of London says there is 'no reason to be alarmed!'"
His comment appeared to refer to an earlier statement by Mr Khan that: "Londoners will see an increased police presence today and over the course of the next few days. There's no reason to be alarmed."
Mr Khan's spokesperson replied to the barb by saying the mayor "has more important things to do" than respond to an "ill-informed tweet" that deliberately took his comments out of context.
Asked about Mr Khan's continued criticism of the president, Mr Giuliani said: "Many of the people who go to London are American citizens. Half of us at least – or more, this president is more popular than ever – support the president. Even more people support (the office of) 'the president'."
"I think it's highly inappropriate and he is probably suffering from some of the criticisms that Trump has made of him, which are valid.
"Now if crime's gone up, he's not paying attention to his job. This is one mayor who's telling him how to reduce crime but you have to pay attention to the job to do that."
Mr Trump is expected to make his first visit to the UK as president in July. It will be be a low-key working trip, rather than a state visit.
Mr Khan recently has said he would "be more than happy to meet with President Trump", despite what he has said about "diversity, about those who follow my faith, [and] about London", in order to show him London's spirit.
Mr Trump was due to open the new US embassy in London in February, but cancelled the trip, saying the building was too expensive and tweeting that he was not a "big fan" of the decision to move its location.
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The prospect of Mr Trump visiting the UK has seen backlash and the promise of protests and rallies from some of the British public.
Previously, more than 1.8 million Britons signed a petition demanding that the president be prevented from making a state visit to the UK.