The train travelling from Niagara Falls to Toronto crashed near Burlington, operator Via Rail said.
About 75 people are believed to have been on the train, and 45 were injured. It is not clear what caused the accident.
Images from the scene showed at least one carriage lying on its side.
Via Rail said the three people killed were employees, and a fourth crew member suffered minor injuries.
TV footage showed the seriously injured being carried away on boards and stretchers while others were led out of the wreckage by emergency workers.
Three passengers were airlifted to hospital, one with a heart attack, officials said.
Burlington Mayor, Rick Goldring, said the crash had caused damage to nearby buildings.
Passenger Deanna Villela, of Welland, Ontario, said she felt a slight bump before the train left the tracks, sending people and luggage flying.
Canada's Transportation Safety Board said it had launched an investigation.
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