Label bosses and VMG Salsoul have accused the star of using horns and strings from Salsoul Orchestra's 1975 hit 'Chicago Bus Stop (Ooh, I Love It)', TMZ reports.
The lawsuit claims that Madonna never paid to use the sample in the song, from which she has made huge profits since its release in 1990.
The alleged sampling was only recently discovered by VMG Salsoul using new technology designed to isolate and identify specific sounds.
VMG Salsoul, who are claiming copyright infringement, are demanding unspecified damages.
Madonna is currently on her world tour, which comes to London's Hyde Park on July 17.
Compare The Salsoul's 'Chicago Bus Stop' and Madonna's 'Vogue' below: