Sadiq Khan will replace the European Union flag that is flown outside City Hall with an image designed by artist David Shrigley for the 'London is Open' campaign.
The EU flag will be taken down from City Hall at 11pm on Friday evening, a spokesman for Mr Khan told SE1.
It will be replaced by the 'London is Open' David Shrigley flag which will be flown alongside the Union Flag.
"It is more important than ever that we promote the message of London as a welcoming, open city," said the mayor's spokesman.
"Our city will always continue to look outwards, and trade and engage with the entire world – including with the European Union."
Pro-Brexit members of the London Assembly have been urging the Mayor to take down the EU flag since the 2016 referendum.
Earlier this month Peter Whittle AM, of the Brexit Alliance, demanded at Mayor's Question Time that Mr Khan "get it down and put it in the dustbin".
For the latest local news and events direct to your inbox every Monday, you need our weekly email newsletter SE1 Direct.
7,000+ locals read it every week. Can you afford to miss out?
Read the latest issue before signing up