Initial results show North Carolina has approved a constitutional amendment effectively banning same-sex marriage or civil unions.
State election officials said 61% had voted for the measure banning gay unions, and 39% opposed it.
Recent polling had suggested the ban would pass, defining marriage as only between a man and a woman.
North Carolina law already bans gay marriage but the vote enshrines this in the constitution.
Recent comments in favour of gay marriage by US Vice-President Joe Biden had reignited debate over the issue.
‘Laws of nature‘
President Barack Obama has not taken a position on gay marriage, describing his views as “evolving”.
Days before the primary, high-profile supporters on both sides attempted to make their views heard.
Former President Bill Clinton recorded phone messages urging voters to reject the amendment.
Meanwhile, a statement in support of the ban by the Reverend Billy Graham appeared in 14 North Carolina newspapers over the weekend.
National groups poured money into campaigning on the issue, with the anti-gay National Organization for Marriage donating a large chunk of the $1.5m (£932,000) raised by ban supporters Vote for Marriage.
The North Carolina group against the amendment, Coalition to Protect NC Families, raised $2.5m. Its largest single donor was the LGBT advocacy group Human Rights Campaign.
Light to moderate turnout was reported across the state on primary day.
Joe Easterling, who described himself as a devout Christian, told the Associated Press he voted for the amendment.
“I think it is important that the state of North Carolina’s laws are compatible with the laws of nature but, more importantly, with the laws of God,” Mr Easterling said.
Another supporter told the Charlotte Observer: “Our church has taken a stand on this.”
Linda Toanone, an opponent of the amendment, argued that “everybody should have the same rights as everyone else. If you’re gay, lesbian, straight – whatever”.
With the passage of the amendment, North Carolina becomes the 29th state to use its state constitution to explicitly ban gay unions.
While including a ban in the constitution would make it more difficult to overturn than the existing law, one state legislator said changing times would eventually undermine the measure.
“It’s a generational issue,” State House Speaker Thom Tillis told a student group at North Carolina State University in March. “If it passes, I think it will be repealed within 20 years.”
Eight states and the District of Columbia allow gay and lesbian couples to marry. Same-sex marriage laws have passed this year in New Jersey, Maryland and Washington state.
New Jersey governor Chris Christie vetoed his state’s law, while opponents have promised challenges to Maryland and Washington’s laws.
The North Carolina vote came shortly after Mr Biden said he was “absolutely comfortable” with same-sex married couples having the same rights as other married couples.
On Monday, Education Secretary Arne Duncan also spoke of his unequivocal support for same-sex marriage.
Mr Obama has previously backed gay rights and civil unions but has stopped short of supporting marriage.
White House press secretary Jay Carney said on Tuesday that Mr Obama’s positions were “consistent with his belief that it is wrong to take actions that would deny rights to LGBT citizens or rescind rights already provided”.
The president faces increasing pressure from advocacy groups and from donors to his re-election campaign, many of whom see gay rights as a key issue.