The historical and artistic events of the Sanctuary are closely linked to its devotional character. At the end of the 14th century, Franciscan Tertiaries built an oratory here to contain the panel painting bearing the image of the Madonna. History and legend combine as regards the origins of the painting; according to popular tradition it arrived miraculously from Greece in 1345 and was carried to the top of the mountain by a crippled shepherd. The small church, which was built with donations from the town’s citizens, soon became inadequate for the great flux of devotees. 

The Archbishop of Pisa therefore decided to entrust it to the Jesuati monks who set about extending the church in 1445. At that time the sanctuary consisted of a single rectangular hall with a longitudinal development, ending with the marble altar by Silvio Cosini (1530), now kept in the sacristy. When the Jesuati were suppressed in 1668, Montenero was handed over to the Theatines who gave the sanctuary its present appearance. The paintings in the elliptical atrium are the work of the converted Theatine, Filippo Maria Galletti. They draw their inspiration from the Episodes from the Life of Mary and are placed within a decorative context that symbolically evokes the garden as a metaphor of Paradise. 

The interior, with its marked Baroque influence, is adorned with stucco decorations and a profusion of polychrome marbles. The six side altars commemorate the Societies that contributed financially to the building of Montenero. A stucco cornice runs along the aisle, supported by the chapel arches and the buttresses. In the rooms between the windows there are further canvases by Galletti portraying Episodes from the Lives of Joseph and Mary. The wooden ceiling, designed and carved in 1680 by the Pisan, Pietro Giambelli, bears three large paintings on canvas. These show episodes from the life of St Jerome of Thiene, founder of the Theatines, and are again the work of Galletti. In 1721 work began on the construction of a new chapel intended to provide a worthy setting for the Holy Image. A cruciform body was added to the church, giving it a traditional Latin cross plan. 

The project was initially given to the architect Giovanni Del Fantasia who had to abandon the work because he was also involved in the construction of the Church of Santa Caterina in Livorno. The sculptural decoration was therefore entrusted to Giovanni Baratta from Carrara, and when he died he left the task of the splendid Gloria for the main altar to his grandson Giovanni Antonio Cybei. The tabernacle contains the panel of the Madonna of Montenero generally attributed to the Pisan artist, Jacopo di Michele, known as Gera, and active in the second half of the 14th century. 

The decoration of the dome (1771-74) is by the Florentine Giuliano Trabellesi who dealt with the theme of the Coronation of Mary according to traditional iconography. The fresco draws on the illusionist features of 18th-century painting, filtering them through a new classical survey of detail. A rich and varied palette allows the artist to give exuberance to the representation, while still respecting a harmonious balance of tone. The pendentives of the dome feature the four Evangelists in gold mosaic by Traballesi, while the Livornese, Giuseppe Maria Terreni painted the architectural decorations of the tambour with floral motives, putti and allegorical symbols of the virtues of the Virgin. The side chapels contain a long gallery of votive offerings bearing witness to the Livornese people’s devotion to the Madonna and their veneration of the Madonna delle Grazie. 

In 1783 the Grand Duke Pietro Leopoldo abolished all religious confraternities, including the Theatines, leaving the care of the sanctuary to three secular priests. It soon became clear, however, that they were not suited for the task. When the Grand Duchy passed to Ferdinando III in 1792, the Benedictine monks of Vallombrosa decided to take over the care of the church and to rescue it from its state of neglect. The Vallombrosans continued to take care of the sanctuary, except during the Napoleonic period when they were sent away. The sanctuary is still in their hands today.