The railway was built by the Società Pio Central (Italian for Central Pius Company), named in honour of Pope Pius IX, who had overturned the Vatican's previous opposition to innovations such as railways in the Papal States.
It was headed by the haulage contractor Mr. Hubert Debrousse, a French engineer, and 950 workers were engaged on 27 railway work sites.
The railway equipment consisted of eleven French steam locomotives (built by Cail & C. of Paris), one English steam locomotive, 21 first class passenger carriages and 25 second class passenger carriages.
On 2 July 1860 the permanent Civitavecchia station opened for service and construction began on the section of the line from Civitavecchia to the Papal States' northern border with Tuscany, which was inaugurated and opened for service on 22 June 1867.
On 22 October 1863, the line was extended south over the Tiber on a lift bridge—built out of cast iron and prefabricated in England—in order to connect with the new Roma Termini station.