The death of the bison was similar to that of Sauron, the 660-kilo (1,455-pound) male whose decapitated body was found nearly a week earlier at the reserve.
A spokesman for the central government's representative in the eastern Valencia region where the private Valdeserrillas reserve is located said the animal had been decapitated after death.
"Either it died of a natural death, or it was poisoned before being decapitated by an axe."
The European bison, the continent's largest wild land mammal, once roamed across most of the continent but it was severely hunted until it finally became extinct in the wild in 1927, according to the World Wildlife Fund.
Neither of the dead animals had any bullet wounds, and reserve spokesman Rodolfo Navarro told AFP earlier this week he thought that Sauron might have been poisoned and decapitated by hunters who wanted its head as "a trophy".
At the time, another three animals belonging to the same herd were missing, and staff feared they could have been sick or frightened and hiding.
The second bison to have been discovered dead was one of those three.
The two other animals have been found safe and sound.
Police have opened a probe into the deaths.
The owners of the reserve plan to introduce female bison to the site later this year to allow the herd to reproduce.
The animal has been reintroduced into the wild across Europe over the past few decades and the species' global population now stands at around 5,500, according to the environmental organisation.