This dissertation examines images of ambiguously gendered human bodies in early modern Italian art (1600-1750). Specifically, it explores how artists rendered bodies that underwent a physical change, transforming from conforming men and women into ambiguous or gender-fluid entities. However, the alterations to these figures’ forms did not relegate them to the period’s third category of gender, the hermaphrodite. Rather, they entered into liminal spaces between the defined boundaries of male and female. Focusing on Italy and its interactions with other European centers, including England and Spain, I explore the ways in which artists constructed a new visual language for the portrayal of ambiguously gendered bodies by turning to a variety of novel sources. In particular, I examine artists’ use of medical knowledge from treatises on congenital diseases, anatomical illustrations, and surgical manuals. In combination with artists’ use of classicizing myths and religious doctrines, these medical sources enabled artists to render figures as recognizable derivations from the natural order, while still retaining attributes of their humanity. Three case studies demonstrate how these issues manifested on the painted surface: Jusepe de Ribera’s portrait of Magdalena Ventura and Her Husband (1631); Jacopo Amigoni’s Musical Portrait Group: The Singer Farinelli and Friends (1750–1752); and Giovanni Andrea Coppola’s altarpiece Martyrdom of Saint Agatha (1650). The subjects of these paintings—hirsutes (figures whose hair covers the entire body or face), castrati (male singers who were castrated before reaching puberty so that their voices would remain at a prepubescent height and pitch), and Saint Agatha (an early Christian virgin martyr whose breasts were amputated) demonstrate how slippages between conforming male and female bodies existed across early modern life and belief. Drawing from the fields of Art History, Social History, Gender Studies, History of Medicine, and Literature, this dissertation elucidates the early modern preoccupation with understanding bodily difference—a preoccupation, I argue, of equal importance for artists, philosophers, and medico-philosophers as studying and representing the ideal Renaissance body. This project, therefore, presents an opportunity to reconsider the parallels between early modern definitions of non-conforming bodies and issues surrounding gender identity in contemporary society.