The motion was voted down with a show of hands from delegates, a result that is a boost for Jeremy Corbyn.
But the circumstances of the vote will likely see the row over Labour’s Brexit policy rumble on.
There were calls for a card vote from some in the hall – a formal ballot which is held at Labour conferences when votes are seen to be close.
However, these calls were in vain and the proceedings moved on.
There was earlier another boost for Mr Corbyn, as members backed his Brexit strategy to remain neutral on a second referendum until after a general election.
Delegates broke out into a chorus of “oh Jeremy Corbyn” after the result was announced.
But shadow Brexit secretary Sir Keir Starmer insisted it was “highly likely” Labour would end up supporting Remain anyway.
Mr Corbyn wants to strike a new deal with Brussels within three months of coming to power and then decide which side to back in an ensuing public vote at that point.
But he had come under pressure to adopt a pro-Remain stance now, with a number of Labour frontbenchers calling for a decisive break with its current policy.
The push has caused a furious row, with claims the party has been trying to stifle debate and that those pro-Remain Labour frontbenchers have been disloyal and guilty of undermining their leader.
Union boss Len McCluskey told Sky News on Sunday that Labour frontbenchers should back their leader on Brexit – or “step aside” from the shadow cabinet.
Reacting to Monday’s developments, he told Sky’s Jon Craig: “It’s been settled that people want to support Jeremy and support the NEC statement and go forward.”
Mr Corbyn’s stance was set out in a statement from the party’s ruling National Executive Committee on Saturday.
The NEC endorsed it without a formal meeting, despite opposition from some members.
This sparked claims the party was trying to shut down debate on the issue.
Jon Lansman, boss of the Corbyn-supporting group Momentum, said before the votes that the process had been a “travesty”.
Shadow cabinet ministers Emily Thornberry and Tom Watson are among those who have been calling for the party to adopt a pro-Remain stance, as well as London mayor Sadiq Khan.
“I believe we must strive night and day, whatever it takes, to keep Britain in the European Union,” Ms Thornberry told the conference earlier.
But their appeals have fallen on deaf ears, for now at least.
There was confusion at the conference as the pro-Remain motion was voted on.
As delegates cast their votes, chairwoman Wendy Nichols could seen having a discussion with the party’s general secretary, Jennie Formby.
Announcing the result, Ms Nichols said: “Sorry I thought it was one way… and Jennie said something else, so.
“Yes, that was lost.”
But there was further confusion and commotion, with Ms Nichols again reiterating that the motion was rejected, despite appeals for a formal vote from some members.
Mr McCluskey told Sky News it was “absolute nonsense” to suggest that Ms Formby overruled Ms Nichols on the vote.
Michael Chessum, national organiser for Another Europe is Possible, said Labour members would be “deeply disappointed” by the decision.
“It is possible that the Remain motion had a majority in the CLPs [Constituency Labour Parties], but because there was no card vote we will never know.”
“Though it is not the policy we supported, the calling of a special conference to democratically decide Labour’s Brexit policy was a concession which we won.
“But a fudge is not a unity position. It is deeply divisive among members.”
Shadow Brexit secretary Sir Keir Starmer, who used his conference speech to say he would be backing Remain, said he was “disappointed” at the result.
At a conference fringe event he said: “I don’t think there is a deal that’s going to be as good as the deal we’ve got [by staying in the EU].”
Conservative Party chairman James Cleverly said: “Jeremy Corbyn and Labour have once again dodged making any decision on Brexit, proving yet again all they offer is more of the same – more talk, more indecision and more pointless delay.
“It is now official Labour policy to delay Brexit until at least 2020 and even longer if the EU demand it.”
Lib Dem leader Jo Swinson, who has committed her party to cancelling Brexit if it wins power, accused Mr Corbyn of displaying a “total lack of leadership”.
“Jeremy Corbyn has repeatedly had the opportunity to put the full force of the Labour behind a Remain position, but he has once again shown today that he is a Brexiteer at heart,” she said.
The SNP’s Europe and foreign affairs spokesman Stephen Gethins said: “Jeremy Corbyn is stuck firmly on the fence, refusing to come down – and today’s votes show the rest of the Labour Party is happy to stay there.
“This is a real abdication of leadership.”