Sadiq Khan has accused John Cleese of acting like Basil Fawlty after the actor said London is "not really an English city".
The Mayor Of London said that Cleese is acting as if still inhabiting his famous Fawlty Towers character.
"These comments make John Cleese sound like he's in character as Basil Fawlty," Mr Khan said.
"Londoners know that our diversity is our greatest strength.
"We are proudly the English capital, a European city and a global hub."
Mr Khan's comments came after Cleese wrote to his 5.6 million Twitter followers that friends had confirmed his previous opinion that London was no longer English, and noted the city's strong pro-Remain stance in the Brexit referendum.
Cleese tweeted: "Some years ago I opined that London was not really an English city any more.
"Since then, virtually all my friends from abroad have confirmed my observation
"So there must be some truth in it...
"I note also that London was the UK city that voted most strongly to remain in the EU."
The Twitter post drew a strong reaction from users condemning his comments, and questioning what it meant for a city to be English.
Author Caitlin Moran was among the people commenting on his post, writing in response to his tweet: "John Cleese is in the process of moving to Nevis, in the Caribbean.
"I hope there's a (sic) old Nevisian comedian ready to moan about how Charlestown won't 'really be a Nevisian town any more.' Goddamn immigrants."
Writer James Felton raised a similar point.
Sherlock star Amanda Abbington tweeted: "What's happened to John Cleese...?"
While TV presenter Rick Edwards wrote: "Just when you think you can't love John Cleese any more!! It turns out you can't.
The official Leave.EU account praised Cleese, writing: "Bravo to British comedy legend and Brexiteer @JohnCleese for speaking up about the state of London The liberal luvvies on Twitter are in meltdown over his refusal to apologise for telling the truth!".
In response to the criticism Cleese said Twitter users had misunderstood him, and denied that his claims about London were linked to race, calling these accusations "projections".
He wrote: "I suspect I should apologise for my affection for the Englishness of my upbringing, but in some ways I found it calmer, more polite, more humorous, less tabloid, and less money-oriented than the one that is replacing it."