Four of the men are under criminal investigation on the mainland and the fifth has said he is "assisting" authorities there.
All five worked for Hong Kong's Mighty Current publishing house, known for often salacious titles critical of the Beijing government.
Publishing associations, booksellers and advocacy groups from the United States and Europe penned the joint letter to Hong Kong's leader Leung Chun-ying, saying the case sent a "terrifying message" to the semi-autonomous city's literary world.
"This case has sent a terrifying message to writers, publishers, and booksellers in Hong Kong that tackling politically sensitive topics can imperil an individual's freedom and safety," the letter, dated February 17 and published in full on PEN American Center's website, said.
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"These developments offer chilling confirmation of the steady erosion of freedom of expression in Hong Kong," it added.
The letter urged Hong Kong "to do everything in its power to investigate these cases and demand the release" of the men.
Last week the UK issued its strongest comments yet over the case of Lee Bo -- the only one of the five publishers to have disappeared from Hong Kong. Lee is a British passport holder and was last seen at a Hong Kong book warehouse in December.
British foreign secretary Philip Hammond said Lee had been "involuntarily removed" from Hong Kong in a "serious breach" of the joint agreement to protect the city's freedoms, signed before the handover of the city from Britain to China in 1997.
The Chinese authorities have not said Lee is under investigation.
In letters purportedly written by him, Lee has said he is on the mainland helping with investigations.
Beijing hit back at Hammond, slamming London for making "groundless accusations against China" and meddling in Chinese domestic affairs.