Chuka Umunna has left Labour's shadow cabinet by "mutual agreement", citing "key differences on policy" with new leader Jeremy Corbyn.
Mr Umunna said in a statement he could not see himself campaigning to leave the European Union but Mr Corbyn "made it clear... he does not wholeheartedly share this view".
Mr Corbyn is currently finalising his first shadow cabinet.
Labour's deputy leader Tom Watson has urged MPs to back Mr Corbyn.
But Yvette Cooper, Liz Kendall, Tristram Hunt, Rachel Reeves, Chris Leslie, Jamie Reed, Emma Reynolds and Shabana Mahmood have already said they will not serve in Mr Corbyn's shadow cabinet.
John McDonnell, a key ally of Mr Corbyn who has been tipped as a possible shadow chancellor, predicted the new shadow cabinet would be "broad based" - and said an announcement would be made shortly.
In other developments:
Mr Umunna described leaving the shadow cabinet as one of the most difficult "personal political decisions" he has had to make.
"Whilst there is much on which Jeremy and I agree, there are a number of key points of difference on policy which I believe it would be dishonest to deny exist," he said.
He said he would "find it difficult to abide by the collective responsibility" of being in the shadow cabinet - "not least on the European referendum".
Mr Corbyn has said he is not content with the EU as it stands, but wants to stay to fight for a "better Europe". He had previously refused to rule out campaigning to leave.
Mr Umunna added: "It is my view that we should support the UK remaining a member of the EU, notwithstanding the outcome of any renegotiation by the prime minister, and I cannot envisage any circumstances where I would be campaigning alongside those who would argue for us to leave - Jeremy has made it clear to me that he does not wholeheartedly share this view."
Few MPs backed Mr Corbyn in the leadership contest - which he won with 59% of the vote - but his deputy Mr Watson told the BBC's Andrew Marr Show there was "zero chance of a coup".
He did however concede that he had differences with Mr Corbyn over the leader's desire to scrap the Trident nuclear deterrent and take the UK out of Nato.
But he said his message for those refusing to serve in the shadow cabinet was "there's always someone else that can do a front bench job".