European Commission chief Jean-Claude Juncker said Friday that "deserters" from the EU would have to accept being outsiders, in his strongest warning yet against Britain voting to leave the bloc next month.
"Deserters won't be welcomed with open arms," he told French newspaper Le Monde.
"The United Kingdom will have to accept being considered a third party, who we won't be bending over backwards for," he said.
"That is not a threat, but our relations will no longer be as they are today".
Juncker's intervention comes after US President Barack Obama used a visit to London last month to urge Britain not to leave the European Union when it votes in a referendum on June 23 and after warnings from the International Monetary Fund and the Bank of England of the economic risks of going it alone.
Prime Minister David Cameron has also warned of the economic and security risks of leaving the bloc, but opponents insist Britain will thrive.
Justice Secretary Michael Gove, a leading Brexit campaigner, has conceded that Britain may no longer have access to the single market but says it will be in other EU countries' interests to maintain strong trade ties.
In a speech last month, Gove denied that Britain was "destined for a bleak, impoverished future on the outside".
"What will enrage, and disorientate, EU elites is the UK's success outside the union," he said.
There was no immediate reaction to Juncker's comments from the "Leave" camp, but he is not a popular figure in eurosceptic circles, viewed as the man behind a push towards greater EU integration.
Former London mayor Boris Johnson, a leading figure in the Brexit campaign, sparked outrage last weekend by saying the EU was behaving like Adolf Hitler in trying to create a European superstate.
With just over a month to go, the latest opinion polls suggest the "Remain" camp is in the lead.
A compilation of the six latest polls by the What UK Thinks research project puts "Remain" on 55 percent and "Leave" on 45 percent, excluding undecideds.
Some of Britain's leading actors, including Benedict Cumberbatch, Keira Knightley, Patrick Stewart, Helena Bonham Carter and Chiwetel Ejiofor, entered the debate on Friday with a warning against a "leap into the unknown".
In a joint letter, 282 people from the worlds of film, music and literature cited William Shakespeare and David Bowie to claim that British creativity "inspires and influences the rest of the world".
"We believe that being part of the EU bolsters Britain's leading role on the world stage. Let's not become an outsider shouting from the wings," they said.
However, the "Vote Leave" campaign was dismissive, describing the intervention on Twitter as "multi-millionaire actors unaffected by uncontrolled EU migration lecturing British people".