Geelong train passengers are being warned of industrial action, which could cause big delays on Thursday morning.
Operations staff including conductors, train controllers, station and customer service staff will be taking part, but drivers will not.
The action will be held between 3am and 7am Thursday, which will bring the network to a halt during and after the stopwork.
They are fighting for better pay and conditions.
Vik Sharma from the Rail, Tram and Bus Union warned action would continue until a deal was reached.
“Our members are fighting for their job security and conditions,” he said.
“Technilogical advancements in the railway poses a serious risk to those frontline jobs and we want to protect those jobs.”
However V/Line boss Matt Carrick said he was frustrated the union had chosen the path again, following action in December.
“We’ve been negotiating in good faith and we’re disappionted the strike is taking place,” he said.
“While there has been some progress in discussions, this action doesn’t help.”
Mr Carrick said the union needed to adjust its demands.
Passengers are being urged to plan their day.