Begun in 1583 at the order of Grand Duke Francesco I de' Medici, who commissioned the project of Bernardo Buontalenti, the Palace was planned to replace the more ancient Medicean Palace (today headquarters of the Prefecture). Built by encapsulating existing buildings such as the thirteenth-century Tower of the Verga d'Oro, which had belonged to the Gaetani family, and the Cantone Tower, still visible, the building was remodeled several times from the 17th to the 19th century. The Residence of the Court during its sojourns in Pisa, it hosted famous scientists such as Galileo Galilei, who illustrated his astronomical discoveries from the medieval tower of the Verga d'Oro, and Francesco Redi, who died there on March 1, 1697.

The National Museum, set up in 1989 in the halls of the Palace, whose aspect remains that of a Court residence, displays numerous works of art (tapestries, portraits, paintings of various subject and historic coats of armour) that belonged to the Medici, the Lorraine and the Savoia families.