The accident at 3:30 pm (2030 GMT) saw one locomotive and five cars come off the tracks, on the route between Niagara Falls and Toronto, said VIA Rail.
There were 75 people on board plus a crew of four at the time of the crash, VIA Rail said.
The three killed were all VIA Rail crew members, the company confirmed.
Following reports of people trapped after the crash, the company said all passengers had been removed from the train.
"Emergency services were on the scene shortly after it occurred and are attending to those requiring their assistance," added VIA, saying three passengers were airlifted to hospital with serious injuries, and 42 other passengers and one crew member were also taken to local hospitals.
Canada's Transportation Safety Board said earlier Sunday it was launching an immediate investigation into the accident.
