MPs and peers have published a letter in The Daily Telegraph expressing their opposition to gay marriage.
Culture Secretary Maria Miller outlined the Government's plans on allowing same-sex marriage last week. A bill is expected to come before Parliament at the end of January.
The Conservative leadership faces strong oppostion, however, as nearly 60 members of the Commons and Lords laid down their challenge in the letter.
Signatories include David Burrowes, David Davis, Fiona Bruce, Jim Dobbin, and former Archbishop of Canterbury Lord Carey.
The letter states that the Government has "no mandate" to redefine the meaning of marriage.
"We recognise the value of a loving and committed relationship and we respect civil partnership, but affirm the distinctive value of marriage reflecting the complementarity of a man and woman often evidenced in parenthood," it reads.
"At the last election, none of the three main parties stood on a platform to redefine marriage. It was not contained in any of their manifestos, nor did it feature in the Coalition's Programme for Government. These facts alone should have led to extreme caution on the part of those calling for this change to be made."
The politicians go on to accuse the Government of "ignoring" the "overwhelming" public response against the plans.
They refer to a petition calling for the traditional definition of marriage to be upheld, which was signed by more than 500,000 people.
"The consultation has ignored the views of 500,000 British residents in favour of anonymous submissions from anyone anywhere in the world," the letter continues.
"We believe that the Government does not have a mandate to redefine marriage."
Miller told the Commons last week that a "quadruple lock" would be put in place to ensure that discrimination claims are not brought against churches that refuse to host gay marriages.
However, the letter in The Daily Telegraph raises concerns about the freedom of people who oppose gay marriage, and expresses doubts about legal guarantees for freedom of conscience.
"We will be seeking legal guarantees of the same freedom of conscience for our constituents and religious organisations to teach, preach and express a traditional view of marriage," the politicians said.
"We are sceptical that the proposed protections will prevent the erosion of liberties of religion and conscience."