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303 Gallery was first established by owner and director Lisa Spellman in 1984 at 303 Park Avenue South. In addition to its address, the gallery name also references Alfred Stieglitz's "Intimate Gallery" artists-collaborative located in Room 303 of the Anderson Galleries building. Spellman's 303 Gallery moved to the East Village in 1986 where she invited such artists as Christopher Wool and Robert Gober to curate special artist projects and collaboratives. 1989 marked 303 Gallery's move to 89 Greene Street in Soho where Vito Acconci, Andreas Gursky, and Rirkrit Tiravanija had solo exhibitions. It was on Greene Street that Spellman initially exhibited with Doug Aitken, Hans-Peter Feldmann, Rodney Graham, Karen Kilimnik, and Collier Schorr. In 1996 303 Gallery was among the first galleries to move to Chelsea, opening at 525 West 22nd Street. In 2008, Spellman purchased a new building at 547 West 21st Street, the gallery's current home.

This May, 303 Gallery will move to new location at 507 West 24th Street, designed by principal architect StudioMDA with Murdock Solon Architects.