A first occupation of the area started only in the early sixteenth century on the impulse of the Count of Conversano Andrea Matteo III Acquaviva d'Aragona. He allowed about forty peasant families from Noci to settle here and cultivate the land, with the obligation to give him the tenth of the crops.

In 1635 his successor, Count Giangirolamo II (1600–1665) erected an inn with a tavern and an oratory and started the urbanization of the forest with the construction of few small houses. The expansion of the urban area was helped by the abundance of limestone, karst and calcareous sedimentary, and by the permission of the count to build houses only with dry walls without the use of mortar, which would become the peculiar trulli. This obligation to have houses built with dry stones was an expedient of the count to avoid paying taxes to the Spanish viceroy of the Kingdom of Naples. The centre of Alberobello was built on the streets of the ancient river Cana, where is now the largo Giuseppe Martelotta.

Alberobello remained a fief of the Acquaviva of Aragon until 27 May 1797, when King Ferdinand IV of Bourbon elevated the small village to the royal city, freeing it from the feudal servitude of the counts. On 22 June 1797, the first mayor Francesco Giuseppe Lippolis was elected.

Alberobello is the only inhabited center with an entire district of trulli, and is considered to be the cultural capital of the trulli of the Itria Valley.