The train, owned by MinRes, left the mine yesterday bound for Esperance carrying 7488 tonnes of iron ore fines.
The company will start stockpiling ore at the port ahead of the first shipment, expected within four weeks.
The Southern Port Authority collaborated with MinRes to modify the rail car dumper at Esperance to be capable of unloading the company's fleet of bottom-dump rail wagons.
MinRes only acquired the mine from Cleveland-Cliffs in late August, restarting production shortly after.
MinRes managing director Chris Ellison said the first train was a significant milestone for the company, as well as the people of Esperance and the Yilgarn.
"When we decided earlier this year to take on the Koolyanobbing operations previously operated by Cleveland-Cliffs Inc, we did so because of our firm belief we could sustain a viable iron ore export operation in the region and safeguard hundreds of jobs in regional Western Australia," he said.
"This successful outcome would not have been possible without the great assistance of Premier Mark McGowan and his government, the leadership team at the Esperance port and Paul Larsen and his team at Arc Infrastructure.
"We look forward to building up our iron ore stockpiles at the port of Esperance ahead of loading our first ship before the end of this year."
Koolyanobbing produced 92,000t of iron ore in the September quarter.
MinRes expects the mine to produce 6-6.25 million tonnes of iron ore per annum.
Shares in MinRes closed at A$15.35 yesterday, valuing the company at $2.9 billion.