The region around Ostuni has been inhabited since the Stone age. The town is reputed to have been originally established by the Messapii, a pre-classic tribe, and destroyed by Hannibal during the Punic Wars. It was then re-built by the Greeks, the name Ostuni deriving from the Greek Astu néon ("new town").

Sacked after the fall of the Western Roman Empire, in 996 AD the town became part of the Norman County of Lecce. In this period started the development of medieval town around the summit of the 229 m high hill, there was built a castle (now only parts of the original castle remain) and city walls with four gates.[2] From 1300 to 1463 it was part of the Principality of Taranto and from 1507 (together with what is now the frazione of Villanova and Grottaglie) passed to Isabella, Duchess of Bari, wife of Gian Galeazzo Sforza, Duke of Milan. Under the influence of Isabella, Ostuni enjoyed a golden-age within the wider panorama of the Italian Renaissance. In this period Isabella gave protection to humanists and people of art and letters, including bishop Giovanni Bovio. Isabella died in 1524 and Ostuni passed as dowry to her daughter Bona Sforza, wife-to-be of Sigismund I of Poland, King of Poland. During Bona Sforza's government, Ostuni continued to enjoyed a liberal and magnanimous regime. In 1539 she had towers built along all the shoreline, as protection against anticipated attacks from the Turks who controlled the Balkans. These towers (still existing, including Pozzella Tower, the Pylon, Villanova and others), were permanently garrisoned and communicated through fire beacons.