Number 10 slapped down Adam Afriyie's plan to table an amendment to legislation paving the way for a promised vote in 2017, insisting it would not be allowed to pass "in any circumstances".
"The PM will not let it stand," a spokesman said.
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The Windsor MP said the public was "not convinced" that the Prime Minister would stick to his pledge of a vote if the Conservatives win the general election.
Writing in a national newspaper, Mr Afriyie said delaying posed "significant dangers", including building support for the UK Independence Party (Ukip), a serious concern for many colleagues and activists.
And he claimed the support of "many MPs across all the main parties" for an early referendum.
But he was warned by the fellow Tory MP attempting to steer the leadership-backed legislation through Parliament that the move would delay and even "kill" his private member's bill altogether.
James Wharton said: "This amendment would make it far more difficult to navigate the challenging procedural hurdles we need to overcome and I hope its sponsors might rethink their approach.
"We need to build as broad a base of support for the bill as we can if we are to get it through Parliament."
Meanwhile, Deputy Prime Minister Nick Clegg will say the EU referendum promise is "deeply flawed and bound to unravel." In one of his most strident attacks yet, the LibDem leader will say no renegotiation could ever satisfy Tory eurosceptics hell bent on an exit from Brussels that would be "economic suicide" for the UK and derail the recovery.