Sir Keir Starmer has suffered a major rebellion over his stance on the Israel-Gaza war, with 56 of his MPs voting for an immediate ceasefire.
Jess Phillips, Afzal Khan and Yasmin Qureshi were among shadow ministers who quit their roles to back the motion from the SNP.
Ten of the party's frontbenchers have left their jobs over the vote, including eight shadow minsters.
Sir Keir has instead backed pauses in the conflict to deliver aid.
Announcing she was quitting her role as shadow domestic violence minister, Ms Phillips said she was voting with "my constituents, my head, and my heart".
"I can see no route where the current military action does anything but put at risk the hope of peace and security for anyone in the region now and in the future," she added.
Ms Phillips, Mr Khan and Ms Qureshi, along with Paula Barker, announced they would be leaving shadow ministerial positions in the run-up to the vote.
Sir Keir had signalled before the vote that MPs holding such a role would be sacked if they backed the ceasefire call.
Other frontbenchers Sarah Owen, Rachel Hopkins, Naz Shah and Andy Slaughter have also left their roles after voting for the motion. Dan Carden and Mary Foy left posts as parliamentary aides.
In a statement after the vote, Sir Keir said he regretted the vote of some of his party.
"I regret that some colleagues felt unable to support the position tonight. But I wanted to be clear about where I stood, and where I will stand".
He said Israel had suffered "its worst terrorist attack in a single day" at the hands of Hamas on 7 October.
"No government would allow the capability and intent to repeat such an attack to go unchallenged," he added.
The vote was on an SNP amendment to a government motion on its plans for the year ahead, presented in the King's Speech last week.
It called for an end to the "collective punishment of the Palestinian people" and urged "all parties to agree to an immediate ceasefire".
It was defeated by 125 votes to 294, with the 56 Labour rebels joining other opposition parties to demand a ceasefire, against the Conservatives who opposed it.
There are 29 Labour MPs in the shadow cabinet, but around half of the party's 198 MPs hold some kind of frontbench position, including party whips.
Among the Labour MPs voting in favour of a ceasefire was Stella Creasy, who told BBC Radio 4's Today programme that, while she respected Sir Keir's position, she defied party instruction as a matter of principle.
"Nobody is under any illusions that a single vote in the UK parliament is going to change the situation on the ground," she said, but "advocating for a ceasefire is far better than the alternative of being silent."
SNP Westminster leader Stephen Flynn said it was "shameful that a majority of Tory and Labour MPs blocked calls for a ceasefire - and have condoned the continued bombardment of Gaza".
The voting took place amid demonstrations from pro-Palestinian supporters, who chanted "ceasefire now" outside Parliament.
The UK has seen a series of protest marches demanding a ceasefire in recent weeks, with an estimated 300,000 people taking part in a rally over the weekend, the biggest in the UK since the war began.
In a bid to defuse the ongoing row over the party's position, the Labour leader had tabled his own amendment spelling out his position, which was defeated - but garnered 160 Labour votes.
It supported Israel's right to self-defence after Hamas's "horrific terrorist attack" on 7 October, in which 1,200 people were killed, and called for the release of more than 200 people taken hostage.
But it also said there had been "far too many deaths of innocent civilians and children" since Israel began striking Gaza in response.
The Hamas-run health ministry says more than 11,000 people have been killed in Gaza since then - of whom more than 4,500 were children.
The amendment also called for longer humanitarian pauses to allow aid, calling this a "necessary step to an enduring cessation of fighting as soon as possible".
Sir Keir has argued that a ceasefire would not be appropriate, because it would freeze the conflict and embolden Hamas.
Labour, like the Conservative government, the United States and the European Union, is calling for "humanitarian pauses" to help aid reach Gaza.
Compared with a formal ceasefire, these pauses tend to last for short periods of time, sometimes just a few hours.
They are implemented with the aim of providing humanitarian support only, as opposed to achieving long-term political solutions.
Last week, the US said Israel would begin to implement daily four-hour military pauses in areas of northern Gaza.
There had been intense efforts to minimise frontbench resignations by strengthening criticism of Israel's conduct of the campaign in Labour's own motion.
There will be relief in Labour leader's office that no one who sits round the very top shadow cabinet table broke ranks to support the SNP's ceasefire motion- though they are now looking for eight more junior shadow ministers and two parliamentary aides.
While the rebellion stretched beyond Labour's left wing, the party leadership believe the scale of disunity won't be replicated in other policy areas.
The assessment is that the passion and pressures relating to the Middle East are unique.
Insiders say that Sir Keir's call for a pause not a ceasefire keeps him in lock-step with the EU and US.
But some of his closest allies frankly recognise that calls for a ceasefire from an opposition Labour leader will have no effect on Israeli PM Benjamin Netanyahu, never mind Hamas in Gaza.
So in that sense, there's little logic to calling for it.
But it means politically, he will have to face down continued pressure domestically to change position.