The singer's manager, Caresse Norman, said: "The music was stolen and was not intended for release for several months.
The pregnant superstar's record company has threatened legal action against Napster, whose program allows users to search other users' PCs for MP3 files to download via the internet, amid allegations that the song was "stolen".
The offending material is the title track from Madonna's new album Music, which is not due to come out until the autumn.
The singer's manager, Caresse Norman, said: "The music was stolen and was not intended for release for several months.
"It is still a work in progress. Ultimately those sites that offered a download of Madonna's music are violating her rights as an artist."
Her record company Warner Bros also issued a statement warning of legal action unless the single was taken off Napster.
It read: "Any site that does not remove our copyrighted material runs the risk of civil and criminal prosecution.
Unauthorised
"We expect that the owners who have included this material on their site will comply immediately with our legitimate request that they cease permitting unauthorised downloads of this song."
Madonna is the biggest name to join the ongoing fight over the protection of music copyrights on the internet.
Rock band Metallica is suing Napster for copyright infringement and racketeering, arguing that its software encourages users to freely trade their songs without permission.
Last month the group successfully blocked more than 300,000 fans from using the MP3 site after handing in their names to Napster's US headquarters.
Rap stars Dr Dre and Eminem are also unhappy with the company and Madonna's considerable presence is sure to boost the campaign.
Napster, which counts Public Enemy's Chuck D among its supporters, has said in the past that it is not doing anything illegal because it does not directly provide the copyrighted music.
Blame the labels
The case has prompted reaction from elsewhere in the internet music world.
Simon Bazalgette, of online webcaster Music Choice, said the big labels were already uncomfortable with the rapidly developing technology.
"Napster will only make them more uncomfortable and make them want to put their heads back in their shell," he told BBC News Online.
"If music has been stolen then the artists aren't getting paid and at the end of the day there is going to be less new music which doesn't help anyone."
But David Phillips, of digital download provider icrunch.com, blames the large music labels for ignoring the clear demand for digital music.
"The artists are at the short end of the stick but they should point the finger at the record labels because of their slowness in taking advantage of the digital medium," he said.
"We will see more of it. The demand is producing this piracy."