Labour was accused of being in turmoil after 50 MPs rebelled against the party leader to back a call for the UK to stay in the European single market and customs union, against party policy.
Three frontbenchers who supported the amendment to the Queen’s Speech were sacked, while another resigned.
Daniel Zeichner, the MP for Cambridge and former shadow transport secretary, said it was clear that “any diversion from the published line” will no longer be tolerated by the party leadership.
Labour whips had made clear shadow ministers should abstain on an amendment tabled by former shadow business secretary Chuka Umunna.
The party has carved out a hard Brexit stance despite denying the government a majority at the general election in large part thanks to the votes of young people, who were also overwhelmingly in favour of staying in the EU.
Labour says it wants to pursue a “jobs first Brexit” and rejects the government’s plans for a Repeal Bill to replace EU law with British regulations, but has largely the same stance on seeking tariff-free access to the single market as the Conservatives.
Mr Zeichner said: “I actually think it shows the Labour frontbench is very serious about returning to power.
“In the past people with my views have been tolerated, but today it was very clear that any diversion from the published line would not be tolerated.
“That’s why I resigned, because I felt I had to represent the views of Cambridge, which in my view is not only to stay in the single market and the customs union, but also to remain in the European Union.”
Mr Zeichner added that he supported shadow Brexit secretary Keir Starmer’s approach to EU talks, but had to respect the views of his constituents in Thursday’s vote.
“It’s no surprise there are tensions,” the MP said. “This is not easy politics, when you’re trying to balance these conflicting views from across the country.”
Supporters of the Jeremy Corbyn rounded on the rebels accusing them of indulging in the “worst kind of gesture politics”, undermining party unity.
Union boss Dave Prentis said it was “totally inappropriate” for MPs to “create” a split over Europe just as the Government was coming under pressure over public sector pay.
“Parliament was just seven votes shy of overturning the Government’s cruel pay cap, yet instead of keeping up the pressure, some seem determined to let the Conservatives off the hook,” the Unison general secretary said.
“Undoubtedly Europe is and will remain an important issue, but when unity is so important in the Labour Party, it is utterly self-defeating to become bogged down in the worst kind of gesture politics.”
Labour deputy leader Tom Watson said the vote had been “unnecessary” and that he was “very disappointed” with Mr Umunna for moving the amendment.
“To break away like that is politically unhelpful at a time when the entire Labour Party is buzzing because we did far better in the general election,” he said.
The amendment was defeated by 322 votes to 101 and was supported by the SNP and Liberal Democrats. Mr Umunna said the fight against a “hard Brexit” would continue.
“What it shows is that pro-European backbench MPs of all parties are not going to simply submit to a hard Brexit,” he said.
“With a hung parliament, we have a real chance in future to shape Britain’s exit from the EU to protect jobs and working people.”