Mitt Romney told the Liberty University commencement that marriage is an “enduring” institution that’s reserved for one man and one woman.
The crowd cheered his comments, made days after Democratic President Barack Obama embraced same-sex marriage.
Mitt Romney also said that culture – “what you believe, how you live, what you value – it matters”.
“Marriage is a relationship between one man and one woman,” he said.
Mitt Romney, a Mormon by religion, was given a standing ovation.
Barack Obama, fighting for re-election as president in November, announced his support for gay marriage this week.
It was seen as a politically risky move, especially in the South, where one in three swing voters strongly opposes allowing gays and lesbians to wed.
Virginia is regarded as a key battleground in November.
Addressing the graduation ceremony at Liberty University, Mitt Romney avoided talking about his own faith but stressed the importance of Christian values in American society.
He has so far struggled to gain support from evangelical Christians in his campaign for the Republican ticket.
“There is no greater force for good in the nation than Christian conscience in action,” he told the audience.
While Mitt Romney opposes gay marriage, he has said that same-sex couples should have some rights, including the ability to adopt children.
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