Witnesses say the armed man ran away after a busing the woman for about 30 minutes, and robbing other passengers.
The crime has shocked the country, becoming a topic of discussion on social media.
The rape of a tourist in late March had already raised concerns about security ahead of the football World Cup next year and the Olympics in 2016.
Days later, seven people died as a packed bus crashed off a bridge following an alleged argument between the driver and a passenger.
After these episodes, Rio de Janeiro's mayor, Eduardo Paes, vowed to tighten security and checks on public transport.
'On drugs'
But Friday afternoon's attack comes as a fresh blow, correspondents say.
Witnesses told the authorities the assailant took a group of about 10 passengers to the front of the vehicle, telling the driver to keep on driving.
After taking their belongings, the man is said to have hit a 30-year-old woman with his gun before raping her.
The man, who witnesses said appeared to be under the influence of drugs, then got off the bus and ran across a busy motorway.
Video footage of the bus is being analysed and a sketch of the suspect is being drawn up, police said.
Robberies are common on buses in Rio de Janeiro and other Brazilian cities but the violence and audacity of these attacks has shocked local media.
Curbing violence is a major priority for city authorities ahead of the major global sporting events.
-BB
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