Emergency services were called to Meadowhall Way, near Meadowhall Shopping Centre, at about 7.10pm on Saturday (Jan 30) to reports of a crash.
Police confirmed on Sunday that two men, aged 20 and 24, died in the incident.
Pictures from the scene show a broken rail which it is believed the car crashed through before landing in the River Don.
A heavy police presence remains in the area as investigators seek to establish what happened.
The identities of the men who died have not yet been released.
A South Yorkshire Police spokesman said: “At 7.10pm last night police received reports that a vehicle had entered the river off Meadowhall Way in Sheffield.
“Two men aged 20 and 24 were taken to Northern General Hospital, where they were sadly pronounced dead.
"Family members have been informed and are being supported by specially trained police officers.”
The force also cleared up online speculation the vehicle was being pursued by police at the time.
The spokesman said: "The vehicle involved in this incident was not being pursued or followed by the police."
They added: "It was a very distressing incident for all involved.
"All emergency services were at the scene within minutes and entered the water to help.
"South Yorkshire Police will be releasing further details later."