The trains will be ferrying passengers from Phnom Penh to the southwestern seaport of Sihanoukville over a nine-day trial lasting until April 17 -- a week that coincides with the Khmer new year holiday.
If the service proves popular, the passenger trains could start operating the nearly 270 kilometre route regularly, according to Royal Railway Cambodia, which in recent years has only used the line for freight.
"This is a fantastic occasion. You can see that the carriages are just about full," Royal Railways CEO officer John Guiry told AFP as passengers climbed on board the train in Phnom Penh's station.
"Hopefully, if nothing else...We take some cars off the road and reduce the traffic jams and the frustrations of trying to have a good weekend," he added.
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The carriages were filled mostly with local holidaymakers, who peered outside as the train rumbled through farmland and passed along the scenic coast.
"I'm so excited to be here," he told AFP from inside the train car, decked with the country's flag.
"For Cambodian people we are proud that the train has been put in place once again.