The EU Commission president said he "welcomes" the clarity provided by Theresa May's landmark speech on her Brexit plans yesterday, saying he was ready to work together for a "balanced solution".
But implied he would only believe her claims once Article 50 has been triggered.
Mr Juncker also issued a thinly veiled swipe at US President-elect Donald Trump, telling a meeting of the EU Parliament in Strasbourg that people who want to see the Brussels project fall apart are "wrong".
Jean-Claude Juncker said he want the best possible result from the Brexit talks
He made his remarks straight after an eye-brow raising speech from Maltese prime minister Joseph Muscat, in which the tiny island's chief branded Mrs May's plans for Brexit "detached from reality".
In contrast Mr Juncker struck a more conciliatory tone, but he could not resist getting a dig in at Mr Trump and eurosceptic leaders such as Ukip's Nigel Farage, who he accused of being false prophets.
The EU chief was responding to Theresa May's plan for Brexit
He told the chamber: "Ambition is something we need more than ever to find a good way of reacting to the UK's decision to leave the EU and to prepare for a relationship with Donald Trump in the United States.
"I welcome what the Prime Minister of the UK said yesterday, but I said yesterday a speech alone cannot trigger negotiations. Once the UK has activated Article 50 the negotiations will start and they should be concluded within two years.
"The negotiations are going to be of great significance to that country but also to the 27 member states and I will do everything so that this negotiation ends with a balanced solution while fully respecting our rules."
He also ripped into eurosceptic leaders across the continent, accusing them of having no answers to the problems facing Europe such as the ongoing migrant crisis and sluggish economic growth.
Earlier Maltese PM Joseph Muscat described Mrs May's Brexit speech as 'detached from reality'
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Juncker's many signs of affection
Wed, January 18, 2017
Jean-Claude Juncker is a Luxembourgish politician. Since 2014, Juncker has been President of the European Commission, which is the European Union Executive Branch.
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I will do everything to ensure that the negotiations will be according to the rules and will yield good results
He raged: "Most of our citizens and you and the Commission would have liked Europe to react more rapidly and with greater solidarity to the events of 2016.
"Quite often we end up going down the route of the extremists when we defend our own interests because they make Europe responsible for all ills.
"They are wrong and they fool those people who think that if you close in on yourself and you close your doors to migrants that is the way to solve all problems.
"We need to show those people who think that this is the time to deconstruct, to let it fall apart, we need to show them that they are wrong.
"On their own no country will be able to organise the economy fight unemployment welcome in migrants and fight terrorism. Stand united and strengthened and face all the challenges."
Mr Juncker's response came after Mrs May gave an historic speech in London yesterday lunchtime pledging to pull Britain out of the Single Market and the European Court of Justice (ECJ).
The PM said that people had voted to take back control of the UK's borders and promised a 'Global Britain', with the Government actively pursuing the signing of trade deals with the world's biggest nations.
But that policy was mocked by Mr Muscat during his firebrand speech yesterday, in which he insisted that there is no chance that the UK will end up leaving the European Union with a deal which is equivalent to or better than its current membership terms.
Malta, the EU's smallest nation, yesterday took up the six-month rotating presidency of the Brussels bloc and is set to play a key role in the opening of the Brexit talks, which will start when Mrs May triggers Article 50 by the end of March.