Shepparton Sergeant Trent Buscall said police were called to the scene at the railway intersection on Wren St, Toolamba about 6.45 am.
The driver of the car, an 18-year-old Arcadia man, was flown to Melbourne with “non-life threatening injuries”, Sgt Buscall said.
No train passengers or V/Line staff were injured in the collision.
An Ambulance Victoria spokesperson said the man was treated by paramedics at the scene before he was transferred by helicopter to the Alfred Hospital with serious injuries.
The spokesperson said he was in a stable condition.
A V/Line spokesperson identified the 6.24am passenger service from Shepparton to Southern Cross as the train involved in the incident, and that no injuries to staff or passengers were reported.
Crews were working to assess and repair any damage to the rail infrastructure to services can resume, they said.
The spokesperson said the 25 passengers on board continued their journey on coaches.
“We thank Shepparton line passengers for their patience while we worked to get them to their destination as quickly and safely as possible following an incident at Toolamba,” the spokesperson said.
The spokesperson said V/Line has advised the Office of the National Rail Safety Regulator and will conduct a full investigation into the incident.
V/Line advised passengers to check the V/Line website, app or Twitter for the latest information about services.