Sadiq Khan met Jean Claude Juncker on the eve of 'Brexit Day'
Divorce proceedings between Britain and the bloc will begin on Wednesday when the Prime Minister formally notifies Brussels of the decision to part ways.
Fierce insults have been slung from both sides – with some claiming Britain will be “punished” by EU leaders.
But Mr Juncker sought to bring calm around the proceedings as he insisted Ms May and her Brexit ministers will be welcomed along to orderly negotiations.
The European Commission president told London mayor Sadiq Khan negotiations would occur in a "fair and friendly" way.
The mayor met Mr Juncker during a whirlwind day of talks with EU power-brokers in Brussels.
European Commission President Jean Claude Juncker with Sadiq Khan
here is no need – as some have suggested – for the EU to send a message, or to instil fear, by punishing the UK
A spokeswoman for the commission president said that he conveyed his solidarity with London after the Westminster attack.
He said: "President Juncker also listened with interest to the mayor's views and concerns in the context of the Brexit process.
"He restated that the EU will negotiate in a friendly and fair way."
Mr Khan earlier used a keynote address in Brussels to warn ministers Britain will not stand for being “punished”.
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He said: "Now is the time to be confident in the European Union, and to act with confidence. There is no need – as some have suggested – for the EU to send a message, or to instil fear, by punishing the UK.
"Because a proud, optimistic and confident institution does not secure its future by fear."
Sadiq Khan leads another ANTI-TRUMP parade in London Mon, March 6, 2017 Play slideshow PA 1 of 5
Annie Lennox, Mayor of London Sadiq Khan and Bianca Jagger lead crowds across Tower Bridge during the March4Women event in London, ahead of International Women's Day
The intervention, the day before Theresa May is set to invoke Article 50 and formally trigger Brexit, was intended to put the "emotional" argument for a fair deal between the EU and UK.
Mr Khan used the address to stress that London will remain Europe's only global city after withdrawal, and that both sides need each other to boost their own prosperity.
"My city is not only the beating heart of Britain's economy, but the single most important organ for growth across Europe. I say this with friendship and all due respect – but a bad Brexit deal that hurts London would hurt the European Union too."
Prime Minister Theresa May will officially begin Brexit on March 29, 2017
The mayor called on Mrs May to make a gesture of goodwill when she invokes Article 50 by guaranteeing the rights of some 3.3 million EU citizens in the UK.
He also urged citizens across the EU to push their leaders to conclude a fair deal with Britain that is in everyone's interest.