The bus, carrying 49 people in all, was thrust 30 meters along the track near the small industrial town of Marhanets near Dnipropetrovsk at around 9 a.m.
The Emergencies Ministry said 41 people, two of them children, had died, but officials said the toll could rise further because many survivors were badly injured.
The crumpled hulk of the bus was still trapped under the front of the locomotive several hours after the accident. Television footage showed the bodies of victims laid out under blankets alongside the track.
President Viktor Yanukovich, on a visit to the region at the time, declared Wednesday a day of national mourning.
“A tragic event occurred this morning ... According to preliminary data, about 40 people have been killed,” Prime Minister Mykola Azarov said at the start of a Cabinet meeting.
Ukrainian police said in a statement that the bus, carrying 49 passengers and a driver, was traveling from a hospital in Marhanets to another nearby town, Gorodishche.
Thirty-eight of those who were killed died at the scene of the crash. Nine people were being treated for injuries in hospital.
