Just over 78 per cent of voters in the tiny European Union country said "No" to granting foreigners the vote, according to final results.
Had the vote been carried, the landlocked nation of over half a million people would have been the first in the EU to grant foreign-born residents the right to vote in all the country's elections.
Prime Minister Xavier Bettel had billed the referendum as a chance to boost the democratic credentials of the wealthy duchy, which is nestled between Belgium, France and Germany.
"The message in clear and has been understood. This is not a success for the governing parties," said Bettel. "We will respect the result."
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While campaigning yesterday in the capital, also called Luxembourg, the premier said a "Yes" vote was also a vote for democracy and diversity.
"There is no other European country where only 40 per cent of the population elects its representatives," Bettel told journalists ahead of the referendum, in which 244,382 people were eligible to vote.
About 46 per cent of the total population of 565,000 people are foreigners.
Bettel's Democratic Party, which is in coalition with the Socialists and Greens, had proposed to enfranchise foreigners resident in Luxembourg for over 10 years.
Around 35,000 mostly European migrants met the criteria.
Bettel has led the charge for change in Luxembourg on a number of fronts. He was the first EU leader to enter into a same-sex union when he married his gay partner last month.
"What matters is that we carry on with integration in this country and that we continue to live together and respect one another," Bettel said.