The one-year-old cub was killed in an incident which may have been deliberate, according to Lipu, the wildlife charity that manages the reserve.
“Great sorrow for the animal’s death was followed by anger for the evidently non-natural causes of its demise,” the charity wrote in a statement on Facebook.
A post-mortem confirmed that the wolf was killed by blunt force trauma, likely caused by the impact of a vehicle.
Suspicions were aroused immediately by the absence of marks on the ground to indicate that the driver had braked or swerved before hitting the young animal which, due to a degenerative disease, dragged its hind legs and moved at a slow pace.
Only authorised vehicles are permitted to enter the 180-hectare reserve, located near Fiumicino airport, leading to speculation that the wolf may have been hit by poachers.
The wolf family, including five cubs, made international headlines after being filmed with a hidden camera at Castel di Guido last September.
The wildlife sanctuary kept the litter a secret for months until they were sure of the cubs’ survival.
Wolves are a protected species in Italy since 1971.