The Coalition for Marriage says the Conservative Party's push for gay marriage was a "key factor" in their humiliating Eastleigh by-election loss.
The Tories were relegated to third place by UKIP, while the Lib Dems revelled in a win coming at the end of a week in which the headlines were dominated by the Chris Huhne court case and Lord Rennard scandal.
In 2010, UKIP scored just 3.5% of the vote in Eastleigh but this soared to 27% in Thursday's vote.
Gay marriage has divided the Conservative Party, with more MPs voting against the Bill than for it in the House of Commons last month.
Colin Hart, campaign director of C4M, said UKIP's success was "clear that natural Tory voters are deserting the party over gay marriage".
In the run-up to the by-election, C4M distributed 13,000 leaflets in the area explaining why they believe marriage should not be redefined and clarifying where each of the candidates stood on the issue.
"Voters in Eastleigh are angry that in a troubled economy the Government continues to pursue a marginal issue like gay marriage when living standards for families are falling," said Mr Hart.
"In 2010 none of the three main political parties had redefining marriage in their manifesto. UKIP's stunning second place result in this by-election should serve as a wake-up call to the Prime Minister."