V/Line has been notified by the Australian Rail Tram and Bus Industry Union that operations staff will take industrial action from 3am to 7am on Wednesday, December 13.
Only non-driving grades will participate in the action, including conductors, train controllers, stations and customer service staff and authorised officers.
However, the union says it will bring the entire V/Line network to a halt.
About 93 per cent of its members last week voted in favour of taking action as workers seek a 17 per cent pay rise over four years.
RTBU branch secretary Vik Sharma said the union was focused on minimising the impacts of the action on the public, encouraging commuters to plan their working weeks accordingly.
He said the union would continue to meet with V/Line and negotiate in good faith as members would prefer to avoid the stoppage if their concerns are addressed.
"V/Line needs to stop blindly driving this corporate cowboys' train and listen to its workers," Mr Sharma said in a statement on Monday.
"We make no apologies in our fight to secure decent conditions for our members."
V/Line chief executive officer Matt Carrick urged the union to reconsider, declaring the planned action could disrupt tens of thousands of passengers.
"We'll continue to work with the union to deliver excellent employment conditions for our workforce and a better service for our passengers," he said.
"We will work hard to minimise the impacts of disruption on passengers."
V/Line is finalising replacement coaches and plans to notify passengers of the arrangements as soon as possible.
It comes after almost 18,000 officers began industrial action on Sunday after months of failed pay negotiations with Victoria Police.
The state government also remains locked in protracted pay disputes with the Victorian branches of the United Firefighters Union and Community and Public Sector Union.
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