"This song is not a love song for some of us but for all of us," said Latifah as she introduced the special segment.
When asked by Ryan Seacrest about the groundbreaking event before the show, Madonna stumbled over her words, saying: "I was told not to say anything"
"I mean I wouldn't do any old song with any old artist, but history is going to be made tonight," she finally revealed, while speaking through a gold grill.
Among the new brides was Lewis' sister, Laura, who reportedly said "I do," to boyfriend, Alex, on live TV.
Macklemore & Ryan Lewis Win Best New Artist GRAMMY
After being deputized by Los Angeles County, Latifah officiated the mass nuptials, which included "gay, straight, old, young, of many races and many colors," according to the New York Times.
"Same Love," which features songwriter Lambert on the chorus, overtly addresses the cultural divide on the issue of gay marriage. "A certificate on paper isn't going to solve it all/but it's a damn good place to start," read the lyrics. The title is nominated in the song of the year category at the awards.
In a statement to The Hollywood Reporter, GLAAD called the performance "the latest in a long line of signs that our nation not only accepts, but celebrates the love and commitment of gay couples today."
"When such a critically acclaimed and popular rap artist puts marriage equality center stage at one of the biggest events of the year, it is the latest in a long line of signs that our nation not only accepts, but celebrates the love and commitment of gay couples today," said GLAAD President Sarah Kate Ellis (who is married to Kristen Henderson of the band Antigone Rising). "Macklemore and Ryan Lewis aren't only talented musicians, but are setting the bar high for our allies around the country today."
Earlier in the week, Grammys producer Ken Ehrlich pledged a night of surprises.
"All things are possible on the Grammy Awards," the producer said when previously asked about the Madonna appearance, later adding: "It’s what we do, one of the trademarks of this show, what I’d like to believe separates the Grammys from these other shows. You see artists do things that they just don’t do anywhere else."
Macklemore & Ryan Lewis were nominated for seven honors at the 2014 awards, including album of the year for "The Heist."
They were early winners for best new artist, as well as taking home Grammys for best rap album, best rap performance and best rap song for "Thrift Shop."