Police say Ram Singh hanged himself in Delhi's Tihar jail, but defence lawyers and his family suspect he was murdered.
Home Minister Sushilkumar Shinde described his death as a "major lapse".
Mr Singh, 33, was one of five men being held in the case, which outraged India and sparked debate about the treatment of women. They all deny the charges.
A sixth suspect is being tried by a juvenile court.
Singh's death comes as a huge embarrassment for the authorities who are already under enormous pressure over the case.
"It is a major lapse in security, certainly it is not a small incident. Action will be taken," Mr Shinde is quoted by the Agence France-Presse news agency as saying.
Tihar jail spokesman Sunil Gupta told the BBC that Ram Singh appeared to have hanged himself with an improvised rope made from a blanket at about 05:00 local time on Monday (23:30 GMT Sunday).
He said an inquiry had been ordered and Ram Singh's body would be taken for a post mortem examination later on Monday.
The spokesman denied Indian media reports that Ram Singh had been on suicide watch, and said three people were sharing his prison cell.
Ram Singh's lawyer, VK Anand, told the BBC he was informed about his client's death by police.
"This is shocking news. There were no circumstances to suggest that he would commit suicide. He was happy with the trial, it was going on smoothly," Mr Anand said.
Mr Singh's father, Mangelal Singh said his son had a badly injured hand and could not have hanged himself.
He also said his son had been raped in prison by other inmates and repeatedly threatened by prisoners and guards.
"My son has not committed suicide," he said.
Ram Singh and the four other adult defendants - his brother Mukesh, Pawan Gupta, Vinay Sharma and Akshay Thakur - have been on trial in a fast-track court.
The case was due to resume on Monday. Reporting restrictions mean few details of the trial have been made public.
They face 13 charges, including murder, gang-rape, kidnapping and destruction of evidence. If found guilty they could face the death penalty.
The maximum sentence the juvenile would face if convicted would be three years in a reform facility.
The 23-year-old rape victim, who was not named for legal reasons, was with a male friend when she was attacked on a bus and thrown from the vehicle on 16 December.
She died in a Singapore hospital on 29 December from massive internal injuries.
Ram Singh was accused of being the driver of the bus.
The case provoked nationwide protests and demands for tougher penalties for rapists, as well as greater protection for women.
Tihar prison, which authorities say is the largest in Asia, is India's most high-security prison.
The 55-year-old jail houses more than 12,000 inmates although it has an official capacity of about 6,000.
The complex includes nine separate prison facilities.
Media reports quoting the chief of Tihar prison Vimla Mehra say there were 18 deaths in the jail last year, of which two were cases of suicide.
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