Work on the £50 billion high-speed rail project will begin in 2017 as planned, Transport Secretary Patrick McLoughlin said after a 33-strong Tory revolt in the Commons failed to wreck the scheme.
Former Cabinet minister Cheryl Gillan, who led the rebellion against the HS2 scheme which will cut through Tory heartlands in the Chilterns, indicated that she would keep up the fight by scrutinising the project "inch by inch" as it progressed through Parliament.
Her amendment won the backing of 32 MPs in the lobbies, plus a Conservative teller. The rebels included chairman of the 1922 Committee of Tory backbenchers Graham Brady and former Cabinet minister Caroline Spelman.
Some 13 Labour MPs also backed the amendment proposed by Mrs Gillan, which criticised the HS2 project and particularly ministers' refusal to publish the Major Projects Authority report into the risks of the scheme.
Ahead of the vote a senior Government minister threatened to resign over the scheme unless his constituency was guaranteed sufficient help dealing with the impact.
Europe Minister David Lidington, who missed the Commons vote because he was on an official trip to Estonia, vowed to sacrifice his job in future if he failed to secure adequate mitigation and compensation for the Aylesbury area.
A total of 47 Conservative MPs missed the vote - including Prime Minister David Cameron, a move dubbed "extraordinary" by shadow transport secretary Mary Creagh on Twitter.
In the main second reading vote, MPs voted 452 to 41 to back the principle of the HS2 project, with any serious threat to its progress removed when Labour indicated it would support the legislation.
The first stage of the scheme will link London and Birmingham, with later extensions taking the high-speed route north to Leeds and Manchester.
Mr McLoughlin said the decision on building a new north-south line could not be ducked any longer.
He said: "By voting in favour of the hybrid Bill, Parliament has made a clear commitment to a key part of the Government's long term economic plan.
"HS2 is a once in a generation opportunity to create jobs and develop skills, provide the extra space we need on our rail network for commuters and freight and better connect our biggest cities.
"I am aware of the concerns some who live very close to the HS2 route have. I am confident however that by working together we can ensure this vital new north-south railway is designed in the right way, and we will have spades in the ground in 2017 as planned."
Speaking after the vote, rebel ringleader Mrs Gillan said: "This is a large number of MPs unconvinced that HS2 is the solution to our country's infrastructure problems. Government should realise that this project will be closely scrutinised every step of the way.
"Many colleagues also abstained this evening which shows that the scepticism of this project runs much more deeply than the voting figures suggest."
Mr Lidington was one of several ministerial critics away from the Commons on official business - thus avoiding a whip-defying vote against the Government line that would almost certainly mean leaving the front bench.
Attorney General Dominic Grieve - like Mr Lidington, a Buckinghamshire MP, representing Beaconsfield - also skipped the vote as he had ministerial engagements in Newcastle, which his office said were long-planned.
Andrea Leadsom, a vocal critic of the scheme before being made a Treasury minister in the last reshuffle, was in Brussels.
Mr Lidington told the Bucks Herald newspaper: "I will resign at a later stage of the bill if they don't get mitigation, and that for me includes a Chilterns tunnel."
He said he understood local criticism of his decision not to register his protest in the Second Reading vote.
But he said: "If I stood down I would just be one more MP that is against HS2, but by staying in I have the inside track, it's a pragmatic political judgment."
Mrs Gillan, whose Chesham and Amersham constituency lies along the proposed route, indicated that she wanted Mr Lidington to remain as a minister to fight the plans from within government.
"As it stands, Buckinghamshire will take all the pain and have none of the gain," she said.
"It is important to remember the voices of our Buckinghamshire colleagues in Government are equally as important as backbenchers' voices to speak for us, if not more so. I want allies inside Government as well as on the backbenches as we scrutinise this project."
Tory rebel Michael Fabricant, sacked as a Conservative Party vice-chairman over his opposition to the project, claimed David Cameron had performed a U-turn over the scheme, which was first pushed by Labour transport secretary Lord Adonis.
He said: "Five years ago, the Prime Minister, leader of the Conservative Party, said the Adonis route is profoundly wrong, that its whole implementation would be damaging to the environment, be damaging to local areas which could otherwise enjoy peace and quiet and would be damaging to the nation as a whole.
"And yet here we are five years on with the Government supporting the original Adonis plan. I find that quite extraordinary."
But London Mayor Boris Johnson accused campaigners objecting to HS2 on environmental grounds of talking "bollocks", claiming they only really care about their house prices.
He said protesters "pretend" they are campaigning about woodland or wildlife but what they are "really furious" about is the impact the HS2 proposals are having on property values.
Mr Johnson told Total Politics magazine: "It's bollocks. They're not campaigning for forests, they're not campaigning for butterflies. They pretend to be obviously, but what they're really furious about is that their house prices are getting it."
Later Ms Creagh said: "It is extraordinary that David Cameron couldn't be bothered to turn up to vote.
"Once again, the Prime Minister has shown he is the weak leader of a divided party. He is unable to stand up to rebel ministers opposed to HS2 and unwilling to vote in favour of his own Government's biggest infrastructure project."
Jim Steer, founder of the High Speed Rail Industry Leaders Group, said: "We welcome the resounding cross-party support for HS2 shown at the vote. The strong endorsement is a clear signal that Britain is ready and willing to invest in its future.
"The Government, as well as local communities and companies all over the UK, should now focus on ensuring they are preparing as thoroughly and imaginatively as possible to take advantage of the transformational changes HS2 will bring about."
Richard Houghton, of HS2 opposition group HS2 Action Alliance, said: "The vote was always going to be a 'yes' to HS2, given it was a three-line whip. The telling aspects were that there were so many absences and abstentions. That was not an indication of how unimportant this vote was considered, it was proof that it is of massive importance.
"The abstentions and absences should be taken as non-commitment to HS2. The vote means many MPs do not openly support HS2, not including those who were forced to follow party lines. Those 'no' votes and a considerable number of absentees should be sending signals that HS2 is not going to get an easy ride, and could even become a key election issue."
Stop HS2 campaign manager Joe Rukin said: "What we saw today undermines democracy at its core. We saw MPs willing to vote for a Bill because their party had told them to do so, knowing they did not have all the facts, knowing that a Government report which was critical of HS2 was censored, because it would be harder for them to vote for it if everyone knew the truth.
"Irrespective of what you think of HS2, the fact MPs have said 'We don't know and we don't care' about a commitment to spend well over the official 2011 cost of £50 billion should have everyone worried about the competency of all the MPs who voted for this Bill."
Mr Cameron's official spokesman declined to say what the Prime Minister was doing at the time of the vote.
Asked why the PM did not attend the Commons for the division, the spokesman told a Westminster media briefing: "He doesn't always vote.
"The Prime Minister has very clear and strong support for HS2 and he has made that clear on countless occasions and will continue to do so."