The protected industrial action will take place between 3am and 7am on Thursday, with Rail, Train and Bus Union 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 Department of Transport and Planning said.
"Replacement coaches will not stop at metro train stations," the department statement read.
"Passengers who need to travel should expect significant delays and plan their day accordingly."
Metropolitan, freight and the Sydney-Melbourne XPT train services will still operate during planned industrial action.
Workers have been bargaining with V/Line since June but RTBU branch secretary Vik Sharma said the company was yet to address key concerns.
"In the current climate of automation, major changes and an uncertain economy, members are fighting for what all workers across Victoria deserve – job security and decent conditions," he said last week.
"V/Line continues to drag their feet and seems to be in no rush to reach an agreement."
V/Line chief executive officer Matt Carrick also said talks would go on.
"We strongly encourage the union to work collaboratively with us to reach a fair and reasonable agreement," he said.
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