According to legend, the city of Rome was founded in 753 BC on the banks of the Tiber about 25 kilometres (16 mi) from the sea at Ostia. The island Isola Tiberina in the centre of Rome, between Trastevere and the ancient center, was the site of an important ancient ford and was later bridged. Legend says Rome's founders, the twin brothers Romulus and Remus, were abandoned on its waters, where they were rescued by the she-wolf, Lupa.

The river marked the boundary between the lands of the Etruscans to the west, the Sabines to the east and the Latins to the south. Benito Mussolini, born in Romagna, adjusted the boundary between Tuscany and Emilia-Romagna, so that the springs of the Tiber would lie in Romagna.

The Tiber was critically important to Roman trade and commerce, as ships could reach as far as 100 kilometres (60 mi) upriver; there is evidence that it was used to ship grain from the Val Teverina as long ago as the 5th century BC. It was later used to ship stone, timber and foodstuffs to Rome.

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