The Botanical Garden of Pisa, or "Giardino dei semplici", was instituted near the church of San Vito in 1544 by order of Cosimo I de’ Medici in the intent to collect plants and herbs with medicinal and pharmacological properties, called simplicia. This was the first botanical garden joined to a university structure. The garden layout was entrusted to Luca Ghini, famous botanist from Imola. In 1563, the Garden was transferred to the convent of Santa Marta and, finally in 1591, arrived at its final destination on via Santa Maria. Numerous scientists contributed to its development and fame, including botanists Luca Ghini, Andrea Cesalpino, Giuseppe Casabona, Pietro Nati, Michelangelo and Angelo Attilio Tilli, Giorgio Santi, Gaetano Savi, and Teodoro Caruel.

A gallery of natural and manmade exhibits, a chemistry laboratory and a library (most of which flowed into the University Library) were adjoined to the Garden. Between the late 18th century and the early 19th century, the chemistry laboratory was refurbished, and a greenhouse and tepidarium were built. The building that today houses the Department of Botanical Sciences, the Laboratories and the Herbarium were also built in the 19th century.

In the 16th-18th centuries, the Garden became the location of a veritable "art bottega", capable of producing naturalistic images, often of the highest quality. Manuals of projects for beds and compartments, botanical and zoological tables, botanical waxes, drawings, paintings and engravings of subjects even from outside the natural sciences, were done by artists who worked at the Garden in close contact with scientists. Those who made significant contributions included Daniel Froeschel, an artist who worked at the court of Rudolph II of Hapsburg, Giorgius Dyckman who accompanied Casabona on his trip to Crete, and even Filippo Paladini.

Today, the collection of living plants includes a seed bank, an arboretum, the scientific collection, a section devoted to Mediterranean geophytes, and one dedicated to pharaonic flora, medicinal plants, tropical plants, palms. The collection also contains plants more than one-hundred years old. Didactics, conservation, dissemination and study, constitute the main activities in which the collections are used.

The Garden shares a library and archive with the Botany Museum, housed in the Library of the Department of Botanical Sciences.