The ad, from the online wedding registry Zola, shows two brides exchanging vows before sharing a kiss at the altar.
The family-friendly network, known its heavily-watched holiday romance movies, said the ad was pulled because it was distracting.
"Crown Media Family Networks made the decision to pull the commercials. The debate surrounding these commercials on all sides was distracting from the purpose of our network, which is to provide entertainment value," said Molly Biwer, Hallmark's senior vice president of public affairs and communications.
Biwer confirmed that a conservative group, One Million Moms, part of the American Family Association, had complained about the ads to Bill Abbott, CEO of Crown Media Family Networks, Hallmark's parent company.
The group, dedicated to eradicating "the filth many segments of our society," posted to its website that the Zola commercials made the Hallmark Channel unsafe for family viewing.
Zola had submitted six ads, and four had a lesbian couple. After Hallmark pulled those ads, but not two featuring only opposite-sex couples, Zola pulled its remaining ads, the company said.
"The only difference between the commercials that were flagged and the ones that were approved was that the commercials that did not meet Hallmark's standards included a lesbian couple kissing. Hallmark approved a commercial where a heterosexual couple kissed. All kisses, couples and marriages are equal celebrations of love and we will no longer be advertising on Hallmark," Zola said in a statement.
Hallmark's decision triggered a widespread debate on social media, with #BoycottHallmark trending on Twitter Saturday.
The developments came as Hallmark appeared to be considering more same-sex themed content.
Asked about the possibility of holiday movies based on same-sex relationships, Abbott was quoted in The Hollywood Reporter in mid-November as saying on its TV podcast: "We're open to really any type of movie of any type of relationship."
The Associated Press contributed to this story.