The warning comes after four near misses between trains and vehicles last week.

In one case, a semi-trailer crossed a level crossing despite the fact that the warning lights and bells were operating.

At Colac, a Warrnambool-bound train had a near miss with a black four-wheel drive vehicle.

Less than half an hour later, a V/Line train heading for Traralgon had a near miss with a motor vehicle at the South Gippsland Highway level crossing in Dandenong.

V/Line Chief Executive Officer Theo Taifalos condemned the dangerous and illegal behaviour of motorists.

"This behaviour is extremely disturbing and people need to stop flouting the law before someone is killed or seriously injured," Mr Taifalos said.

"With Victorians heading out of Melbourne for the long weekend and then returning at the end of summer school holidays, V/Line does not want to be involved in a tragic loss of life.

"I urge everybody to be vigilant when approaching level crossings, even if they think they know the train timetable."

Mr Taifalos said safety was his number one priority in the operation of the regional train network.

"Everybody has a right to be safe, including our staff and customers, pedestrians and motorists," he said.

"We work as hard as we can to ensure everybody who interacts with us is always safe, but safety is also a personal responsibility.

"Rail crossing accidents are not only traumatic for survivors and families of people killed, but also for train crew and passengers."

Mr Taifalos said incidents involving near misses between trains and vehicles are always reported to Victoria Police.

As part of V/Line's "Don't push your X-ing luck" safety campaign, motorists are reminded that drivers can be fined $722 and lose four demerit points for entering a railway crossing while a train is approaching.