Nigel Farage was among the British representatives present for the debate, which quickly became heated as Brussels continues to reel from Friday’s result.
Mr Juncker, the President of the European Commission, turned on Ukip MEP Paul Nuttall who clapped after he pledged to respect Britain’s wishes.
“That’s the last time you are applauding here,” he said, to enthusiastic applause from MEPs.
Mr Farage, sitting to the right of Mr Juncker with a Union flag on his desk, laughed during the exchange.
Mr Juncker said he was “sad” at the UK’s decision, adding: “I’m not a robot, I’m a human being.”
“Our British friends have expressed their view by universal suffrage and the majority view of the British people demands respect,” he added. “Democracy is a democracy and we must respect it.”
Before the session opened he was seen in conversation with Mr Farage, who grimaced after receiving a kiss on the cheek.
The European Commission President said the “British remain our friends” as David Cameron prepared for an awkward meeting with EU leaders.
In the first session since the UK voted to leave the 28-nation bloc on June 28, he was due to discuss the implications of the Brexit as market volatility continued.