Gaga has been avoiding the public eye since cancelling her Born This Way Ball world tour to undergo surgery for a labral tear on her right hip in February, which left her temporarily wheelchair-bound.
Waving a rainbow flag in the air, Gaga broke into an acappella rendition of the National Anthem during her surprise on-stage appearance, which came the same day as an appeals court in California ruled that same-sex marriages in the state can begin immediately.
"I'm here tonight to honour my heroes and luckily I have many to look up to - and many to look at tonight ... We want nothing less than every other American," Gaga told the crowd.
"We are not a niche, we are part - a big giant part - of humanity. We are the continuum. We are ongoing. We continue the spirit of the first official Gay rally in 1970 in New York, one year after Stonewall.
"And although this weekend is a time for victory, I am a citizen of New York and I demand the right to safe streets; the violence that has taken place against LGBTs is unacceptable, here and anywhere. Enough is enough."