The petition that was the most-signed government petition since the process was introduced in 2011 and built momentum in the wake of Brexit, or Britain's exit from the European Union (EU), in the June 23 referendum.
However, in an official reply, the UK Foreign Office said this week 33 million people had had their say and "the decision must be respected".
"We must now prepare for the process to exit the EU," it said.
The petition, which was ironically set up by a Brexit supporter before the referendum was held, had called for the government to annul the results if the Remain or Leave vote won by less than 60 per cent on a turnout of less than 75 per cent.
In the wake of the Brexit vote, the petition was shared widely on social media by 'remain' supporters.
British government petitions which reach over 100,000 signatures must be considered for debate in parliament.
"The EU Referendum Act received Royal Assent in December 2015. The Act was scrutinised and debated in Parliament during its passage and agreed by both the House of Commons and the House of Lords," the Foreign Officesaid.
The Act set out the terms under which the referendum would take place, including provisions for setting the date, franchise and the question that would appear on the ballot paper. The Act did not set a threshold for the result or for minimum turnout, it said.
"As the Prime Minister made clear in his statement to the House of Commons on June 27, the referendum was one of the biggest democratic exercises in British history with over 33 million people having their say," the Foreign Officesaid.
"The Prime Minister and Government have been clear that this was a once in a generation vote and, as the Prime Minister has said, the decision must be respected. We must now prepare for the process to exit the EU and the Government is committed to ensuring the best possible outcome for the British people in the negotiations," it said.
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