The female turtle (Rafetus swinhoei) died in Suzhou zoo in southern China.
Experts had tried to artificially inseminate the creature, which was over 90 years old, for a fifth time shortly before she died.
The species has suffered from hunting, overfishing and the destruction of its habitat.
One male, estimated to be more than 100 years old, is left in the Chinese zoo while two other turtles live in the wild in Vietnam. The elusive nature of the turtle means it has been difficult to identify the gender of the pair.
Local staff and international experts had attempted to artificially inseminate the female 24 hours before she died on Saturday afternoon.
They said there were no complications from the operation and she had been in fine health after the procedure, but deteriorated the next day.
The cause of her death is being investigated and the turtle's ovarian tissue was collected for future research.