The measure was passed by 61 percent against 39 percent as of 0230 GMT Wednesday, according to preliminary results from the North Carolina State Board of Elections.
Similar state constitutional amendments have been approved in some 30 US states.
The amendment solidifies and expands already enacted North Carolina law forbidding same-sex marriage.
President Barack Obama has recently come under greater pressure to clarify his position on gay marriage, after Vice President Joe Biden stressed that he personally was fully comfortable with it.
Obama was disappointed that the amendment was adopted, Cameron French, spokesman for his campaign in North Carolina, told US media.
"The President has long opposed divisive and discriminatory efforts to deny rights and benefits to same sex couples," French said. "He believes the North Carolina measure singles out and discriminates against committed gay and lesbian couples, which is why he did not support it."
Money from national interest groups poured into North Carolina ahead of the election -- the National Organization for Marriage which opposes gay unions contributed $425,000 to the Vote for Marriage campaign, according to the latest reports.
The Human Rights Campaign contributed some $257,000 to the opposition, the Coalition to Protect All N.C. Families.
"This overwhelming support for marriage is clearly the reason why President Obama and liberal congressional candidates across the country have not expressed open support for same-sex marriage," Tony Perkins, president of the Family Research Council, said in a statement. "They know that redefining marriage remains a losing position in mainstream American politics."
The Reverend Billy Graham, an evangelical preacher who was born and lives in North Carolina and at 93 remains enormously influential, took out full-page newspaper ads across the state supporting the ban.
"At 93, I never thought we would have to debate the definition of marriage," Graham said in the ads.
"The Bible is clear: God's definition of marriage is between a man and a woman."
On the other side, former president Bill Clinton and his ex-chief of staff, Erskine Bowles, a North Carolina native, recorded telephone messages to voters urging them to oppose the measure. In a statement, Obama's campaign also opposed the amendment.
"The passage of Amendment One is a profound injustice. Singling out a class of citizens for discriminatory treatment is unfair, unlawful and violates basic American values," said Adam Umhoefer with the American Foundation for Equal Rights, a leading US group supporting gay marriage, after the vote.
There are now tens of thousands of same-sex couples in six US states and the District of Columbia who are legally married where they live.
A Pew Research Center Survey last month found that 47 percent of Americans now favor allowing gay and lesbian couples to marry -- well up from 39 percent in 2008 and 31 percent in 2004. Forty-three percent remain opposed.