The Royal Vauxhall Tavern in Lambeth has been listed for its architectural interest and its historic and cultural significance as an "enduring symbol of the confidence of the gay community".
The group that campaigned for it said it was a "fantastic milestone".
But the pub's chief executive said the status could signal its "death as an entertainment venue".
First built in 1860, the Royal Vauxhall Tavern (RVT) hosts alternative, transgender and drag performances and saw Paul O'Grady perform as Lily Savage.
The listing is the first of its kind for a lesbian, gay, bisexual and transgender (LGBT) venue and means its owners must apply for consent before making changes that might affect its special interest.
But the RVT's chief executive, James Lindsay, said he had lost about Â£60,000 of his own money running the venue and the decision to list it was "disappointing" as the RVT in its current form was "not commercially viable".
He said plans to increase the current 380-capacity and modernise the venue would now have to be put on hold.
"I'll do what I can to continue to trade. What is potentially at risk is the whole history of what it was all about - the fringe culture and people coming through and getting their breaks. We have to question whether that will continue," he said.
But the listing has been welcomed elsewhere.
Heritage Minister Tracey Crouch said: "Not only of architectural interest, the venue has a long-standing historic role as a symbol of tolerance and alternative entertainment... I hope that it continues to be filled with laughter and joy."
Amy Lame, chairwoman of the RVT Future group that campaigned for the listing, said: "The listing is a fantastic milestone for our community and a victory against the odds for our beloved pub.
"We look forward to continuing our work to ensure the RVT remains a vibrant space."
Campaign manager Rob Holley added that he hoped the listed status would stop any potential plans to redevelop the site for housing, a growing trend in London.
A well known gay entertainment venue in London has been given Grade II listed status.