The grain truck driver sustained serious injuries in the crash at the Phalps Road level crossing at Pirron Yallock.
The front carriages of the west-bound V-Line train derailed in the crash, but remained upright.
Ambulances, police, State Emergency Services and Country Fire Authority crews attended the scene.
The train driver and 12 passengers received treatment for minor injuries, and an ambulance helicopter took the truck driver to Melbourne for treatment. He was in a serious condition.
V-Line organised replacement buses for passengers to continue their journeys to Warrnambool.
The crash happened minutes after the train left Colac Railway Station.