The droplets of blood belonging to the former pontiff, who is now a saint, were encased in a vial hidden in a golden cross, known as a reliquary, and displayed on an altar dedicated to the Polish pope, who died in 2005 after a 27-year-reign as leader of the Catholic Church.
The theft was discovered by a sacristan who was closing the cathedral, located in the Umbria region, for the night.
Relics typically contain pieces of a saint’s body or part of their clothing and are venerated by Catholics.
The relic was in the Spoleto cathedral temporarily and was due to be moved next month to a new church in Umbria named after the late pope.
Spoleto Cathedral Archbishop Renato Boccardo appealed for the relic to be returned, calling the theft a “grave act”.
“Give the reliquary back to the cathedral and the faithful,” Archbishop Boccardo, who was a close aide to the late pope for years at the Vatican, said in a video message.
“It would be the serious thing to do.”
Archbishop Boccardo said it was not clear if the reliquary had been stolen for ransom, which has happened in the past with other relics in Italy.
Police were reviewing footage of security cameras inside and outside the cathedral.
