"Locomotive CC2207 of SOPAFER-Mali left Kayes this morning at 8:00 a.m., heading for Bamako," Mali's rail line announced on Facebook.
"The departure marks the de-facto resumption of commercial passenger rail traffic between Bamako and Kayes."
After a 400-kilometer trip, the train is expected to arrive at 3:00 am on Saturday.
Service on the line had halted in May 2018 because of poor maintenance, adding to transport problems in a deeply poor country where decent roads are few.
The Bamako-Kayes line is part of a track completed in 1924 during the French colonial era linking Bamako with Dakar, the Senegalese capital, 1,286 kilometres away.
The economy and finance ministry in a statement said that rehabilitation had cost 6.26 billion CFA francs (around $10 million), which would be recouped over the first two years of operation.
Passengers rushed to buy tickets after news that the service had resumed, according to pictures posted by the transport ministry on Twitter.