The protected industrial action will take place between 3am and 7am on Thursday, with Rail, Train and Bus Union (RTBU) members stopping work in their fight for better pay and conditions.
No V/Line trains will run during the stoppage and significant delays are expected throughout the rest of the morning as services gradually return to their normal timetable.
V/Line commuters should avoid travelling on Thursday morning as only limited replacement coaches will be available until 8am.
The coaches will not stop at metro train stations.
Metropolitan, freight and the Sydney-Melbourne XPT train services will still operate during planned industrial action.
"We're focusing on trying to limit the disruptions to passengers," V/Line chief executive officer Matt Carrick told reporters on Wednesday.
"We share the frustration of regional Victorians about this totally unnecessary strike taking place.
"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."
V/Line was committed to expanding rather than decreasing its workforce and the union's claim jobs would be cut was merely "fear mongering", Mr Carrick said.
"There's been talk about the effect of driverless trains and things like that - we're not planning driverless trains," he said.
"It's a really important point that the drivers want nothing to do with this action.
"Progress is being made on an agreement with drivers which demonstrates good faith."
Union members have been fighting since June for job security and decent conditions, RTBU branch secretary Vik Sharma said.
"V/Line continues to drag their feet and seems to be in no rush to reach an agreement," he said last week.
Mr Sharma is set to speak to the media on Wednesday afternoon.
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