First reactions suggest stakeholders are already shaping up for a fresh fight over legislation governing same-sex couples.
After over 99% of the votes were counted, the result was 63.5% against and 36.5% in favour of amendments to the marriage and family relations act that would have instituted full marriage equality for homosexual couples.
Under referendum rules 20% of the electorate or almost 343,000 voters needed to vote against in order to reject the legislation, a quorum that has been exceeded by well over 40,000 votes.
The turnout was 36.2%, which is almost six percentage points more than in a similar vote in 2012, when voters decided on a more limited expansion of the rights of gay couples.
Voters rejected marriage equality in all eight electoral units, with the no vote ranging from 51.4% in Ljubljana Centre to 73% in Ptuj in eastern Slovenia.
Reactions by the groups which joined the referendum campaign indicate the political battle will now be fought over fresh legislation on same-sex couples, though law dictates that legislation running contrary to the vote cannot be passed within a year.
The no camp suggested an attempt would be made to expand the rights of same-sex couples, but not with marriage equality. Instead, efforts will be made to update the existing law on same-sex partnerships.
A bill will be drawn up within a month in a bid to regulate social issues affecting gay couples. The legislation will not include gay adoptions, said Aleš Primc, the co-leader of the Children Are at Stake group, which led the no campaign.
A similar message was delivered by the two centre-right opposition parties, the Democrats (SDS) and New Slovenia (NSi), which see the outcome as a warning to the government not to go too far in expanding the rights of same-sex couples.
France Cukjati, the president of the SDS council, suggested amending the existing law on same-sex partnerships. NSi leader Ljudmila Novak said that "legislation in a different form will be examined once again to finally credibly regulate the status of same-sex couples."
More broadly, the conservative stakeholders said voters protected the traditional family.
Metka Zevnik, the co-leader of Children Are at Stake, said she was pleased that "we protected our children and will continue to be happy mothers and fathers, grandmothers and grandfathers."
The yes camp vowed to continue fighting for marriage equality.
"We will persevere. Human rights are something that needs to be fought for constantly," said MP Violeta Tomič of the United Left (ZL), the opposition party which had proposed the changes to marriage and family relations act earlier this year.
The message was echoed by the partners in the informal coalition bringing together liberal parties from both sides of the aisle.
MP Saša Tabaković of the Modern Centre Party (SMC) said the ruling party "would continue the fight for an inclusive society". He was hopeful a marriage equality bill could still be enacted in the near future.
Likewise, Dejan Levanič of the junior coalition Social Democrats (SD) said a compromise must be found that would secure equal rights for all. He said the party would work to craft a new law.