The fully functioning convenience, dubbed "America", was stolen from Blenheim Palace in Woodstock, near Oxford.
It was one of the star attractions of an exhibition by Italian artist Maurizio Cattelan at the 18th-century stately home, the birthplace of war-time prime minister Winston Churchill and home to the dukes of Marlborough.
During the hearing at Oxford Crown Court in southern England, James Sheen, Michael Jones and Frederick Sines, all in their thirties, pleaded not guilty to stealing the lavatory in the early hours of September 14, 2019.
Bora Guccuk, 40, was not asked to enter a plea over his charge of conspiracy to transfer criminal property.
Visitors to the exhibition at Blenheim Palace, a UNESCO World Heritage Site, were able to book slots to use the precious metal privy but only for three minutes at a time, to limit queues.
More than 100,000 people used the loo during the year it was on display at New York's Guggenheim Museum.
British police said thieves had broken into Blenheim Palace during closing time and made off with the toilet, ripping it from its plumbing and causing "significant damage and flooding."