He was estimated to be about 100 years old.
Officials at Ecuador's Galapagos National Park, where he was living, said the only remaining Pinta Island tortoise was found dead in his corral on Sunday.
A post-mortem will be carried out to determine the cause of his death, the BBC reported.
With no offspring and no known individuals from his subspecies left, Lonesome George became known as the rarest creature in the world.
George was first seen by a Hungarian scientist on the Galapagos island of Pinta in 1972. Since he became part of the Galapagos National Park breeding programme and a symbol of the Galapagos Islands, which attract some 180,000 visitors a year.
Environmentalists tried to get him to reproduce with females from a similar subspecies on the Galapagos Islands, but they failed every time.
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After 15 years of living with a female tortoise from the nearby Wolf volcano, Lonesome George did mate, but the eggs were infertile.
He also shared his corral with tortoises from Espanola island, which are genetically closer to him than those from Wolf volcano, but he failed to mate with them.
While his exact age was not known, Lonesome George was estimated to be about 100, but experts believe the species can live up to an age of 200.
Park officials said that with George's death, the Pinta tortoise subspecies has become extinct. His body may be embalmed to conserve him for future generations, they said.
Tortoises were plentiful on the Galapagos islands until the late 19th century, but were later hunted for their meat by sailors and fishermen to the point of extinction. Their habitat furthermore suffered when goats were introduced from the mainland.
The differences in appearance between tortoises from different Galapagos islands were among the features which helped the British naturalist Charles Darwin formulate his theory of evolution.
Some 20,000 giant tortoises of other subspecies still live on the Galapagos.