The House of Lords has defeated the government to say the referendum on how MPs are elected should only be binding if 40% of the public take part.
Peers, led by Lord Rooker, argued that setting a threshold on turnout would give the result greater legitimacy.
The government, which opposed the move, lost by a single vote. Coalition peers who backed it included Lord Lamont.
There is still uncertainty whether Parliament will pass legislation in time for the poll to be held on 5 May.
The government wants the Parliamentary Voting System and Constituencies Bill to become law by the time Parliament starts its half-term break at the end of next week.
The elections watchdog, the Electoral Commission, has given a warning that if it misses that deadline there will not be enough time to prepare for the referendum.
Monday's defeat was the third that the coalition has suffered on the bill - which will authorise a referendum on whether to replace the existing first-past-the-post system for electing MPs by the alternative vote.
The amendment to the bill - requiring 40% of voters to take part in the poll for it to be binding - was put forward by a former Labour minister, Lord Rooker, who now sits as a Labour Independent.
It was approved by 219 votes to 218 during the "report stage" of the bill.
Lord Rooker said: "A high turnout on a small majority would be as bad as a low turnout with a large majority. I just think before we embark on this we should have an assessment of the result of the referendum."
MPs must now decide whether to retain the proposal or overturn it when the legislation returns to the Commons.
Conservative MP Bill Cash said he was delighed the Lords had taken "a strong stand" - he proposed a similiar amendment to the bill when it was in the Commons, but was defeated by 549 votes to 31.
Labour peers have been fiercely resisting other plans in the bill, which would to cut the number of MPs and redraw constituencies, leading to fears that the legislation will not be approved in time to stage the poll.
Last week peers completed a 17-day long debate on its in-depth committee stage, including one all-night sitting.
The government wants to hold the referendum on changing the voting system on the same day as elections in Scotland, Northern Ireland and Wales and some local elections in England.