The first service set out on Saturday from the the capital Phnom Penh for the tourist areas of the south-west coast.
The service, which will run only on weekends, had a VIP on board - the veteran Prime Minister, Hun Sen.
"The train and railway were almost totally destroyed by war more than 40 years ago," he wrote on Facebook.
"Because our country is now at peace, we have the opportunity to rebuild our transport infrastructure."
The journey from Phnom Penh costs around $7 (Â£4.80) and takes eight hours. It will run to and from the coast once a day from Fridays to Sundays.
The country has hundreds of kilometres of track, but very little is open, after decades of neglect.
Cargo services restarted three years ago, along small stretches of track.
Cambodia has restarted passenger train services on one route after a gap of some 14 years.