V/LINE is urging farmers to mend their fences in the wake of new figures showing a big increase in livestock being hit by trains.
According to the rail operator, 226 animals, including 163 sheep and cattle, were killed by trains last year.
V/Line said it had begun writing to landowners in what it described as the "biggest safety awareness campaign" it had ever conducted.
It said the line between Ballarat and Ararat was the worst stretch, with 42 incidents which killed 125 animals last year, including 105 sheep and cattle.
"Trains hitting livestock is an issue for V/Line, passengers and farmers," V/Line chief executive Rob Barnet said.
"It can delay people, cause damage to our trains and there's also a big cost to the farmer."
Mr Barnet said many farmers had a "low understanding" of their responsibility to maintain fences bordering rail reserves.
"We're keen to raise awareness of the issues in the farming community, to avoid the need for council rangers to impound livestock," he said.
"But we can't allow animals to roam on the lines, so impounding livestock is the next step after we've raised awareness."
