After an emotional debate, US Methodists at a national legislative meeting upheld the denomination's policy that same-sex relationships are "incompatible with Christian teaching".
Delegates at the General Conference in Tampa, Florida voted by about 60% to 40% against softening the language on homosexuality in their Book of Discipline, which contains church laws and doctrine.
The meeting is held once every four years, which means the policy will not come up for a conference vote again until 2016.
Advocates for gay and lesbian Methodists gathered in the convention hall wearing rainbow stoles and protested against the vote by singing and interrupting the meeting.
Some cried when the vote was announced. Methodist leaders briefly shut down business in response to the protest.
With just under eight million US members, the United Methodist Church is the largest mainline Protestant denomination in the country, with a significant and growing membership of more than four million overseas.
However, the number of Methodists is shrinking inside the US, while expanding in African and Asian countries where the church is theologically conservative.
Several overseas delegates spoke against any change in church law on homosexuality, arguing that the Bible forbids same-gender relationships and that homosexuality was not accepted in their countries.
One African delegate, speaking through a translator, compared homosexuality to bestiality.
Methodists have been debating their church stand on homosexuality for four decades.
Other mainline Protestant denominations - including the Episcopal Church, the Evangelical Lutheran Church in America, the Presbyterian Church and the United Church of Christ - have in recent years moved toward accepting gay and lesbian couples.
The United Church of Christ has gone the farthest by affirming gay marriage.