Assembly members have rejected a proposal that same-sex couples should have the right to marry.
The motion was proposed by the Green Party and Sinn Fein.
The DUP had tabled a petition of concern ensuring that the motion would have to command a cross-community majority to succeed.
While 45 assembly members voted to back the move, only three in favour were unionists - UUP MLAs Michael Copeland, Danny Kinahan and Basil McCrea.
Green Party MLA Steven Agnew said he was disappointed the motion had failed, but insisted the "narrow defeat" was a "significant step towards gaining marriage equality for the LGBT community in Northern Ireland".
"There are many who argued, both before and during the debate, that the 'vast majority of people in Northern Ireland' are against marriage equality; however, today's debate puts an end to that argument," he said.
"Close to 50% of elected members of the assembly publicly declared their support for same-sex marriage by voting in favour of this motion today."
Ahead of Monday's debate, the Presbyterian Church had written to all assembly members stating its opposition to any change in the current legal definition of marriage.
In its letter, the Presbyterian Church said it was "not merely an issue of conscience for Christian people and churches, but a very significant one for the whole of society".
It said gay marriage would "effectively demolish generations and centuries of societal norms established on Judaeo-Christian values".
"The steady erosion of such values, with minimal debate about the worldview replacing them, causes us the very greatest concern," it added.
The church argued it was not an equality issue, "as all of the significant legal benefits and rights available through marriage are already equally available through civil partnership".
Westminster is consulting on whether to allow gay couples in England and Wales to marry, while in Scotland the SNP government has announced plans to bring forward a bill on the issue.