The Overland train service between Adelaide and Melbourne is under threat with the South Australian government to end financial support for the service.
The SA backing will cease at the end of December despite ongoing support from the Victorian government.
Great Southern Rail, which operates The Overland, said unless further support was forthcoming the 131-year-old service would end.
Opposition transport spokesman Tom Koutsantonis said The Overland was used by 30,000 people each year and served as a vital link for regional communities.
"This is a vital service, particularly for older South Australians who prefer train travel and for country South Australians who do not live close to airports," Mr Koutsantonis said.
"We all know that once train services cease to operate, they often never return to service."
But the state government said the service had been in decline for a number of years.
"Regional South Australians were the ones voting with their feet to no longer use this service," Transport Minister Stephan Knoll told state parliament on Thursday.
Mr Knoll said over a six-month period this year only 600 people embarked or disembarked from The Overland at a regional station while total passenger numbers had fallen dramatically over the past 15 years.
"Even with it being subsidised we saw a three-fold reduction in the number of people choosing to use The Overland service," he said.
