Nick Clegg indicated today that support for the high speed rail link between London and the north would be a red line in any coalition discussions with Labour at the next election.
The Deputy Prime Minister insisted he would not compromise on support for the controversial HS2 route if the final decision falls after the 2015 vote.
Labour's support for the £50bn scheme has cooled in recent months and Shadow Chancellor Ed Balls has repeatedly insisted there can be no "blank cheque".
MPs will vote tomorrow on a Bill to allow government to spend money planning the route in detail but the project faces many more parliamentary hurdles before work can begin as planned in 2017.
Asked if he would ever compromise on HS2 in a future government, Mr Clegg told reporters at a press conference in Westminster: "No. I was up in Sheffield yesterday talking to business leaders and they are absolutely appalled at the way in which Labour appears to be betraying the north.
"It just beggars belief for a party that constantly parades itself as the authentic voice of the north of England is now prepared to turn its back on the businesses, the communities, the families which I think all the evidence shows will benefit disproportionately from an investment in a high speed north-south railway link.
"I just think it is miserable, it's pathetic that an idea which we inherited from Labour and in all good faith took forward because we thought, given they were the architects of the idea they might support it, that when it becomes politically convenient to play games with it, they start playing games with it.
"I say this obviously with some real fervour as a Sheffield MP because I can see myself that it would just make a dramatic difference to investment in Sheffield and South Yorkshire in, say, the advance manufacturing sector which I think is so important to our local economy in Sheffield if there was a reliable, high capacity, high speed rail link connecting businesses in South Yorkshire with businesses in the financial powerhouses down in London."
Aides later attempted to row back from the comments, insisting that the Liberal Democrats had "not set out any red lines" and calling on Labour to "have the balls" to back it.
The Government yesterday underlined its support for the HS2 even though an official analysis was published showing that the estimated economic benefit of the project is falling.
A detailed 150-page report from the scheme's promoters HS2 Ltd said that the benefit-cost ratio for the full two-phase project is now estimated at 2.3 compared with a figure of 2.5 given in August 2012.
This means that for every £1 spent, the wider economic benefit of the entire scheme will produce a benefit of £2.30 compared with £2.50 estimated last year.
Opponents of the scheme immediately seized on the lower figure as further proof, to them, that the project was a waste of money and should be scrapped.
Mr Clegg said the existing line would be "overwhelmed" quickly if it was simply patched up.
"We need to see this as a great opportunity to heal the north south divide and it is time that Labour get off the fence, stop faffing about playing political games with something which is of national importance and stop betraying the interests of the north of England."