Angalifu, a 44-year-old northern white rhino, was discovered dead early Sunday at San Diego Zoo Safari Park.
The male rhino was receiving veterinary care at the time for a variety of "age-related" conditions, according to the park.
Angalifu's death means there are just five known northern white rhinos in the world – one elderly female at the San Diego Zoo, one at a zoo in Czech Republic, and three others at a national park in Africa.
"Angalifu's death is a tremendous loss to all of us," Randy Rieches, curator of mammals for the San Diego Zoo, said in a statement. "Not only because he was well beloved here at the park, but also because his death brings this wonderful species one step closer to extinction."
The death is a huge setback for northern white rhinoceros, which had staged a minor comeback in the early 1990s.
The white rhino is the second-largest land mammal — only the African elephant ranks larger, according to the World Wildlife Fund.
In 1960, there were 2,000 northern white rhinos, which once roamed southern Chad, the Central African Republic, southwestern Sudan, northeastern Zaire, Democratic Republic of Congo, and northwestern Uganda.
But driven by a lucrative black market for rhino horn, poaching decimated the population. By 1984, just 15 northern white rhinos remained, all of them in Congo's Garamba National Park, according to the WWF.
Under strict protections, the population rebounded to roughly 30 by 1993. But by 2004, the population had shrunk to between 17 and 22. It never recovered.
Angalifu was caught for Khartoum Zoo, Sudan, and was transferred to San Diego in 1990.
Scientists have tried, unsuccessfully, to breed the remaining females. And with the existing northern white rhino population dying out, experts concede the subspecies will almost surely go extinct.
With extinction looming, scientists have been taking samples from the existing stock to preserve their DNA.
In San Diego, semen and testicular tissue from Angalifu have been stored in the "Frozen Zoo" with the hope that new reproductive technologies may allow scientists to recover the species in the future.