His family and lawyer alleged foul play.
"There were no circumstances which could have led to Ram Singh committing suicide. There was no mental stress. He was very happy (about the trial's course)," his lawyer V.K. Anand said.
Singh, 33, had been among five defendants facing the death penalty if convicted of the rape attack, which horrified Indians and set off national protests. A sixth accused is being tried and jailed separately because he is a juvenile.
Singh's death in custody raised further questions about a criminal justice system already under attack for failing to protect the nation's women.
''It's a grave incident," said Shinde, the nation's top law enforcement official. ''It's a major lapse."
The government had ordered a magistrate's inquiry and would take action after it received the report, he said.
Kiran Bedi, the former director of the jail and now an activist, said prison officials had a moral and legal obligation to ensure Singh's safety, and she expressed surprise that authorities had not been monitoring him with cameras.
''You are duty bound to protect the lives of the prisoners," she said.
Mamta Sharma, chair of India's National Commission for Women, said jail authorities had to explain Singh's death ''despite so much protection, so much precaution, so much security."
''This means that even though he was accused of such a heinous crime, the jail administration did not keep a watchful eye on him," she said.
In 2011, 68 inmates in India killed themselves and another eight were killed by fellow inmates, according to India's National Crime Records Bureau. Tihar Jail is badly overcrowded and its 12,000 prisoners are nearly twice as many as it was designed to hold. Bedi said that despite that, the treatment of inmates has improved over the past two decades as the jail became more transparent, with volunteers constantly coming in and prisoners better educated about their rights.
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 had been repeatedly threatened by inmates and guards. Nevertheless, he said he visited his son four days ago and the man appeared fine and gave no hint of any despair that could drive him to take his own life.
Ram Singh also had a badly injured hand and would have been unable to hang himself, his father said, speaking from outside his small home in a New Delhi slum.
"Somebody has killed him," he said, saying he would push for a top-level investigation from India's Central Bureau of Investigation into the death.
Mangelal Singh said he feared for the safety of another son who is also on trial in the rape case.
Vivek Sharma, a lawyer representing another defendant, said he planned to ask the court to provide greater protection for his client.
"In a high-security jail, an occurrence of this kind is highly condemnable. It raises the serious issue of security of the accused persons in the jail," he said.
"My clients don't feel safe in Tihar Jail," said another defense lawyer, A.P. Singh.
Vimla Mehra, the director general of the jail, declined to say how Ram Singh could have managed to kill himself without alerting the other inmates in his small cell or the guards.
"The inquiry is being conducted and it would be premature to make any statement about the details of the incident," she said. Previous reports that Singh was under suicide watch were incorrect a police official said, speaking on condition of anonymity because he was not authorized to release details to the media.
The rape victim and a male friend were attacked after boarding the bus Dec. 16 as they tried to return home after watching a movie, police say. The six men, the only occupants of the private bus, beat the man with a metal bar, raped the woman and used the bar to inflict massive internal injuries to her, police say. The victims were dumped naked on the roadside, and the woman died from her injuries two weeks later in a Singapore hospital.
The attack set off nationwide protests about India's treatment of women and spurred the government to hurry through a package of laws to protect them.
The rape victim's family said that with such a strong case for the prosecution, they had expected Singh to be convicted and executed anyway.
"He knew he was going to get the death penalty, and so he took his life," the victim's brother told the Times Now TV.
Singh's death comes as the trial was deep underway. The four surviving defendants were briefly produced in court Monday.
K.T.S. Tulsi, a former top lawyer in the office of the solicitor general of India, said the suicide should have no impact on the trial, which is being held in a closed courtroom under a gag order that prevents news organizations from publishing details of the proceedings.
He said the death highlighted how important it is for society not to demonize people who have been accused but not convicted of crimes.
"It is so unfortunate that the media goes on to presume that they are guilty and goes on to condemn them and demonize them to an extent that it makes the life of these people not worth living," he said.