The pilot run, which was nearly 100 kilometres, was done without a driver on board, as part of the trials intended to get the company's AutoHaul project up and running by late 2018.
The journey was monitored in real-time by company personnel and representatives of the Office of the National Rail Safety Regulator.
The test, undertaken last week, was from Wombat Junction to Paraburdoo.
Rio Tinto Iron Ore chief executive Chris Salisbury said: “This successful pilot run puts us firmly on track to meet our goal of operating the world’s first fully-autonomous heavy haul, long-distance rail network, which will unlock significant safety and productivity benefits for the business.
“Gains from AutoHaul are already being realised including reduced variability and increased speed across the network, helping to reduce average cycle times."
Rio Tinto runs about 200 locomotives on more than 1700 kilometres of track in the Pilbara, transporting ore from 16 mines to four port terminals.