Marriage between one man and one woman is now the only domestic union recognized under the North Carolina constitution.
In primary elections held Tuesday, North Carolinians voted in favor of Amendment One 60 percent to 40 percent.
Durham County bucked state trends, with 70 percent of voters casting ballots against the amendment.
Although same-sex marriage is already banned under a 1996 law, the amendment will define marriage as between one man and one woman in the state constitution.
Critics have argued that the broad language of the legislation eliminates legal recognition and rights for unmarried heterosexual couples, in addition to banning same-sex marriage, civil unions and domestic partnerships.
The Coalition to Protect All NC Families, a leading anti-amendment group, outspent the pro-amendment Vote for Marriage by nearly $800,000, according to reports by the state Board of Elections.
With the passage of the amendment, North Carolina becomes the 31st state and the last Southern state to pass a constitutional amendment banning same-sex marriage.
In the gubernatorial primaries, Pat McCrory, former mayor of Charlotte, who secured the Republican nomination in a landslide, will face Democratic nominee Walter Dalton, who won the primary with 46 percent of votes.