Starting at 3am on Thursday and running to 7am, Rail, Train and Bus Union members are taking protected industrial action to fight for better pay and job security.
No V/Line trains will run during the stoppage and major delays are expected for the rest of the day.
Limited replacement coaches will be available until 8am but won't stop at metro train stations.
Metropolitan, freight and the Sydney-Melbourne XPT train services will still operate during planned industrial action.
The tens of thousands of V/Line commuters should work from home or make alternative travel arrangements on Thursday morning, RTBU Victorian secretary Vik Sharma said on Wednesday.
"We don't want to create disruption for the commuters but at the same time this is the tool that workers have and we haven't made this decision lightly," he said.
The union and transport corporation have been locked in protracted contract negotiations for seven months with no end in sight.
"They are just not taking this bargaining seriously and we keep ending up in a situation where our members are extremely frustrated and they feel they don't have any other choice."
V/Line workers are concerned the introduction of the new ticketing system would result in job losses, Mr Sharma said.
"Members are concerned that there is a greater risk to the frontline ticket-related roles and therefore we want to enshrine in the enterprise agreement the job security."
V/Line is committed to expanding its workforce and no jobs are at risk of the new ticketing system, chief executive Matt Carrick said, branding the claims as "fear mongering".
"We share the frustration of regional Victorians about this totally unnecessary strike," he said.
"We'll continue to negotiate in good faith to try and get an acceptable outcome, which means that there is an agreement that is fair, that is reasonable and that's affordable."
Mr Sharma said the state government, which will ultimately make the decision, should step in to end the seven-month dispute or face the prospect of more disruption.
"The industrial action is going ahead so if there is a good time for government to intervene, the time, we suppose, is now," he said.
