Tory leader David Cameron has abandoned plans for his promised European referendum after Czech president Vaclav Klaus signed the Lisbon Treaty.
Shadow foreign secretary William Hague said it was "no longer possible" to put the treaty to a popular vote of the British people.
"Now that the treaty is going to become European law and is going to enter into force, that means a referendum can no longer prevent the creation of the president of the European Council, the loss of British national vetoes," he said.
"These things will already have happened and a referendum cannot unwind them or prevent them."
Earlier, Mr Klaus completed the process by formally signing the treaty - ushering in the EU's new rule book after a long ratification process.
Mr Cameron, who had urged Mr Klaus to keep on blocking ratification, found himself isolated when the Czech leader put aside his misgivings and signed the document.
Mr Cameron had hoped Mr Klaus would hold out against the treaty long enough for any incoming Tory government to stage a UK referendum.
This plan was dashed when the Czech president relented and signed much quicker than expected, despite objections to a Czech constitutional court ruling against a claim that the treaty was a recipe for a superstate and incompatible with the Czech constitution.