Siena, like other Tuscan hill towns, was first settled in the time of the Etruscans (c. 900–400 BC) when it was inhabited by a tribe called the Saina. The Etruscans were an advanced people who changed the face of central Italy through their use of irrigation to reclaim previously unfarmable land, and their custom of building their settlements in well-defended hill forts. A Roman town called Saena Julia was founded at the site in the time of the Emperor Augustus. The first document mentioning it dates from AD 70. Some archaeologist assert that Siena was controlled for a period by a Gaulish tribe called the Senones.

The Roman origin accounts for the town's emblem, a she-wolf suckling the infants Romulus and Remus. According to legend, Siena was founded by Senius and Aschius, the two sons of Remus, who was in turn the brother of Romulus, after whom Rome was named. Statues and other artwork depicting a she-wolf suckling the young twins Romulus and Remus can be seen all over the city of Siena. Other etymologies derive the name from the Etruscan family name Saina, the Roman family name Saenii, or the Latin word senex "old" or its derived form seneo "to be old".

Siena did not prosper under Roman rule. It was not sited near any major roads and lacked opportunities for trade. Its insular status meant that Christianity did not penetrate until the 4th century AD, and it was not until the Lombards invaded Siena and the surrounding territory that it knew prosperity. After the Lombard occupation, the old Roman roads of Via Aurelia and the Via Cassia passed through areas exposed to Byzantine raids, so the Lombards rerouted much of their trade between the Lombards' northern possessions and Rome along a more secure road through Siena. Siena prospered as a trading post, and the constant streams of pilgrims passing to and from Rome provided a valuable source of income in the centuries to come.

The oldest aristocratic families in Siena date their line to the Lombards' surrender in 774 to Charlemagne. At this point, the city was inundated with a swarm of Frankish overseers who married into the existing Sienese nobility and left a legacy that can be seen in the abbeys they founded throughout Sienese territory. Feudal power waned however, and by the death of Countess Matilda in 1115 the border territory of the Mark of Tuscia which had been under the control of her family, the Canossa, broke up into several autonomous regions. This ultimately resulted into the creation of the Republic of Siena.
It existed for over four hundred years, from the late 11th century until the year 1555. During the golden age of Siena before the Black death in 1348, the city was home to 50,000 people.

In the Italian War of 1551–1559, the republic was defeated by the rival Duchy of Florence in alliance with the Spanish crown. After 18 months of resistance, Siena surrendered to Spain on 17 April 1555, marking the end of the republic.

The new Spanish King Philip, owing huge sums to the Medici, ceded it (apart a series of coastal fortress annexed to the State of Presidi) to the Grand Duchy of Tuscany, to which it belonged until the unification of Italy in the 19th century.

A Republican government of 700 Sienese families in Montalcino resisted until 1559.

The picturesque city remains an important cultural centre, especially for humanist disciplines.