Sauron, an 800kg bull bison named after a character in Lord of the Rings, was the leader of the herd reintroduced into the wild at the Valdeserrillas Reserve in Valencia, eastern Spain.
But on Friday his decapitated corpse was discovered and he is believed to have been poisoned before his head was hacked off as a trophy.
Three other bison from the herd are still missing and five are suffering the effects of what authorities believe to be poisoning.
A team from Seprona, the environmental crime unit of Spain's Civil Guard have launched an investigation into what they believe is the deliberate poisoning of the herd, either through food or water.
Carlos Alamo, the director of the reserve described Sauron as ““the largest, healthiest and most beautiful specimen” in the herd.
This herd of nine bison bulls arrived at the reserve between June 2015 and May of this year and females were due to be introduced this autumn to start a breeding programme, a project that has now been put on hold.
Bison were only reintroduced back into the wild in Spain in 2010, after becoming extinct throughout much of Europe. The last wild European bison was killed by poachers in 1927, while around 50 remained in zoos.
Herds of bison once roamed the Spanish countryside and were immortalized in cave paintings by stone age man.