Objective: Simonetta Vespucci, considered the most beautiful woman of the Renaissance, is the inspiration and face of one of the most famous paintings of all times, "The Birth of Venus," by Botticelli. She died in 1476 at the age of 23 years. We postulate she suffered from a pituitary-secreting tumor progressing to pituitary apoplexy. The goals of this study were 3-fold: (i) verify that the subject depicted by Botticelli in different paintings represents the same woman; (ii) identify the facial traits affected by the progression of a growth hormone- and prolactin-secreting tumor; and (iii) confirm that the observed changes of the face traits observed in the portraits of Simonetta Vespucci are compatible with the facial traits changes identified earlier. Methods: Comparison among face traits was based on the analysis of the face regions measured by means of fiducial points and their distances, and after pose compensation based on three-dimensional head modelling. Results: In favor of the hypothesis that Simonetta suffered from a pituitary growth hormone- and prolactin-secreting tumor stands changes of her lineaments, a feature which becomes evident over the years and particularly manifest in the Allegorical Lady, where galactorrhea is depicted. Conclusion: We conclude that sufficient evidence is presented to suggest that Simonetta Vespucci, the Venus depicted by Botticelli, suffered from pituitary adenoma secreting prolactin and growth hormon with parasellar expansion. The current interpretation of the Venus strabism should be revisited according to this finding. Abbreviation: GH = growth hormone.