Almost two-thirds of voters said no to a bill that defined marriage as a union between two consenting adults.
Parliament passed a law giving marriage equality in March, but opponents challenged it before any gay couples could marry.
Conservatives were especially opposed to allowing same-sex couples to adopt children.
"This result presents a victory for our children," said Ales Primc from the group Children Are At Stake.
The result demonstrates a cultural split in the EU, where western member states are granting greater rights to gay people but newer central and eastern member states are resisting such moves.
Slovenia`s conservatives were backed by Pope Francis, who called on the mainly Catholic country to "back the family as the structural reference point for the life of society".
But MPs from the United Left party, which initially proposed the change in the law, said the result was a temporary setback.
"It`s not over yet. Sooner or later the law will be accepted," said United Left MP Violeta Tomic.
Slovenia is considered to be among the more liberal former communist countries but gay rights remain a contentious issue there.
In 2012, voters rejected granting more rights to gay couples in a referendum.
Slovenia rejects gay marriage in referendum
21 December 2015
Read: 541
Slovenians have rejected same-sex marriage by a large margin in a referendum.
