An amendment to allow registrars to opt out of conducting same-sex marriages received the support of 150 MPs in a Commons vote.
The MPs many of them expected to be Conservative backbenchers had signed an amendment which if passed would have allowed registrars to opt out of carrying out gay marriages.
The 150 MPs represents a significant opposition to the Government’s plans. But they were defeated in the House of Commons by 340 MPs, meaning a Government majority of 190.
In a second vote, 148 MPs supported an amendment to protect the religious beliefs of a person who believes that marriage is defined as being between a man and a woman. But 349 MPs voted against the amendment, meaning it was defeated by 201 votes.
The MPs opposed to gay marriage had also tabled an amendment which would have allowed teachers and schools to opt out of teaching children about gay marriage on religious grounds.
But a vote was avoided after culture minister Hugh Robertson said he was prepared to review the situation. He said that if necessary, the Government would place amendments in the Lords.
Mr Robertson said: “While I am confident the existing protections (for teachers) are sound, I am very aware of the concerns raised by the Church of England.
“With that in mind I am absolutely able to commit to the House tonight we will take this issue away and discuss with the religious groups who have been engaging in this process and we have been in close contact with all of them.
“We will look at all available means, including an amendment if necessary, to put this issue beyond any doubt in the Lords.”
Despite offering to think again on allowing teachers and schools to opt out of teaching about gay marriage, Mr Robertson insisted the Government could not allow registrars to refuse to conduct same-sex ceremonies.
He added: “I do not believe it is appropriate or right to allow marriage registrars to opt out of conducting same-sex marriages, either permanently or on a transitional basis.
“Like it or not, they are public servants who should carry out the will of Parliament.”
In a third Commons vote, 163 MPs supported an amendment to protect those who opposed gay marriage from discrimination. But 321 MPs voted against the amendment, meaning it was defeated by 158 votes.
Earlier, Tory MP David Burrowes, who proposed the series of major amendments, said the Bill must not become a charter for unfair dismissal of those who do not back the reforms.
The Enfield Southgate MP warned there was a “chill wind” blowing in the country for those who back “traditional marriage”.