Metro has reached agreement with the Rail Tram and Bus Union Loco Division (RTBU LD) regarding the first of the new X'Trapolis trains.
The train will enter service this afternoon at around 4pm at Flinders Street Station, taking passengers for the first time since it was launched on 30 December.
Metro and the RTBU LD met for conciliation at Fair Work Australia on Wednesday, which lead to agreement being reached early this afternoon for the train to enter service.
Metro said it was pleased the issue had been brought to a close.
"We're delighted to have reached a mutually agreeable outcome for all parties, and ultimately it is passengers who are the winners now that the new train is out on the tracks," said CEO Andrew Lezala.
"In order to deliver more than 2,000 services every day and move 400,000 people, we must have access to every train we can get.
"We thank Fair Work Australia Deputy President Ives for his assistance in conciliating this matter," he said.
Public Transport Safety Victoria has also indicated that they have no objections to the train being in service.
The new X'Trapolis will now be timetabled for regular daily service, alongside the 28 other X'Trapolis trains in the fleet.
The second of the 38 new X'Trapolis trains is currently in Ballarat and will be brought to Newport in the coming weeks to commence its testing and commissioning process.