Hox genes act to differentiate and pattern embryonic structures by promoting the proliferation of specific cell types. An exception is Hoxb13, which functions as a proapoptotic and antiproliferative protein during development of the caudal spinal cord and tail vertebrae, and has also been implicated in adult cutaneous wound repair. The adult epidermis, which expresses several Hox genes including Hoxb13, is continually renewed in a program of growth arrest, differentiation, and a specialized form of apoptosis (cornification). Yet little is known about the function(s) of these genes in skin. Based on its role during embryogenesis, Hoxb13 is an attractive candidate to be involved in the regulation of epidermal differentiation. Here we demonstrate that Hoxb13 overexpression, in an adult organotypic skin model, recapitulates actions of Hoxb13 reported in embryonic development. Epidermal cell proliferation is decreased, apoptosis is increased, and excessive terminal differentiation is observed, characterized by enhanced transglutaminase activity and excessive cornified envelope formation. Overexpression of Hoxb13 also produces abnormal phenotypes in the epidermal tissue that resemble pathological features of dysplastic skin diseases. Our results suggest that Hoxb13 functions to promote epidermal differentiation, a critical process for skin regeneration and for the maintenance of normal barrier function.