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Sadiq Khan said the Home Secretary's comments about the policing of pro-Palestinian marches were "incorrect, irresponsible and inflammatory".
London's mayor said the job of senior politicians was to bring communities together and not "stoke division".
Suella Braverman wrote an opinion piece in The Times in which she accused the police of having a softer approach with left-wing protests.
The controversial article was not agreed by Downing Street and officials are looking into what happened.
Mr Khan said the right to protest was one of the "cornerstones of our democracy" but it was important police had operational independence.
"It is not right for politicians to be telling the police which protests they should be banning or allowing," Mr Khan told ITV News.
"Listen to what other are saying whether it is former inspectors of the police, former senior police inspectors, former Conservative politicians.
"We have had this legislation for almost 40 years, the Public Order Act and I have never known any example in the past where Home Secretaries in advance of a protect have been genuinely advocating for that protest to be banned or interfere in police decisions.
It's often the case that senior politicians have conversations with the police privately. What none of us hope to do is interfere with operational matters - it's completely wrong for the Home Secretary to be publicly writing an article [in The Times] that is not only incorrect but inflammatory."
London's mayor said he feared members of the far right would descend on central London this weekend to stoke division.
He added: "It is the case that since the horrors of Hamas on October 7 and since the IDF and Israel went into Gaza and killed many more people there have been protest marches taking place in London.
"The vast majority of the hundreds of thousands of people taking part have been acting in a peaceful, lawful and safe way.
"A small minority have been breaking the law and inciting hatred. The police have made arrests and taken action."
Members of the Tory party have also attempted to distance themselves from what has been described as her "inflammatory rhetoric", with Transport Secretary Mark Harper dodging questions on ITV's Good Morning Britain and actively disagreeing with her on Times Radio this morning. The former chief inspector of constabulary Sir Tom Winsor said Mrs Braverman’s comment “crosses the line” by breaking the convention that a home secretary should not question the operational integrity of the police.
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