The garden was established in 1544 under Cosimo I de' Medici as the first university botanical garden in Europe, and entrusted to the famous botanist Luca Ghini of Imola.

In 1563 the garden was relocated from its original riverside location (now the Medicean Arsenal) to one near the convent of Santa Marta, and in 1591 (under Joseph Goedenhuyze) again moved to its third and current location.

From these early times, the garden has contained a gallery of natural objects (now Pisa's Museo di Storia Naturale), a library (now part of the university library), and portraits of its directors throughout the centuries.

Major collections include herb gardens and arboreta, as well as the old botany institute, built 1591–1595, with a facade ornamented with sea-shells.

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