Emergency services were called to the collision on the Midland Highway, near Bendigo-Murchison Road just after 8am.
Paramedics treated a man in his 60s for upper body injuries who was flown by air ambulance helicopter to The Royal Melbourne Hospital in a serious but stable condition.
A woman in her 40s was taken to Bendigo Health by road for further observation.
Several other people were assessed on scene but were not taken to hospital.
The exact circumstances of the collision are unknown and the investigation is ongoing.
Passenger on the train Paula Huntly described the incident as a bit of a shock.
“There was a big thump and some smoke,” she said.
“We saw the truck and then the front carriage slightly off the track — I was worried the truck was going to explode.”
Mrs Huntly has commended the emergency services who attended the scene.
“V-Line and emergency services moved us off the train, they were fantastic,” she said.
“They got us all replacement buses home.”
In a statement, V/Line said that they have advised the Office of the National Rail Safety Regulator of the incident and will conduct a full investigation.
V/Line CEO Matt Carrick thanked the Echuca line passengers for their patience following the incident.
Anyone who witnessed the collision, with dashcam footage or information is urged to contact Crime Stoppers on 1800 333 000 or online at www.crimestoppersvic.com.au.
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