But his lawyer and family allege he was killed.
Ram Singh, who was accused of driving the bus on which the 23-year-old student was attacked by six men in December, was under suicide watch at New Delhi's Tihar Jail when he hanged himself with his clothes at about 5.30am, officials said.
Singh and four fellow defendants were facing the death penalty if convicted of the attack, which sparked national protests.
A sixth accused is being tried and held separately because he is a juvenile.
India's deputy home minister, RPN Singh, said an inquiry had been ordered into the death.
Ram Singh's lawyer VK Anand said: "There were no circumstances which could have led to Ram Singh committing suicide. There was no mental stress. He was very happy."
Lawyers for the defendants had previously accused police of beating confessions out of the men.
Ram Singh's father, Mangelal Singh, said his son had been raped in prison by other inmates and repeatedly threatened by inmates and guards. Nevertheless, he said he visited his son four days ago when he appeared fine.
Ram Singh, 34, also had a badly injured hand and would have been unable to orchestrate a suicide, his father said, adding: "Somebody has killed him."
