Pannexin (Panx) 1 and Panx3 are integral membrane proteins with sequence homology to the invertebrate gap junctions, innexins, and are expressed in mammalian skin. Panxs form functional single-membrane channels but their importance in regulating cellular function is poorly understood. In this study, the expression of Panx 1 and Panx3 was assessed in mouse skin and the role of these Panxs in kératinocyte differentiation was investigated using differentiation-competent rat epidermal kératinocytes (REKs) engineered to over-express Panxl, Panxl-GFP or Panx3. Endogenous levels of a unique 70 kDa species of Panx3 increased in organotypic epidermis, while Panxl remained unchanged compared to monolayer REKs. Ectopic Panxl, Panxl-GFP and Panx3 predominantly localized to the cell surface in monolayer cultures, but re-localized to intracellular compartments in organotypic epidermis. Furthermore, ectopic Panxl in organotypic epidermis dysregulated cytokeratin 14 expression, disrupted the architecture of the epidermis and reduced the vital layer thickness while ectopic Panx3 had no effect on the epidermis. In summary, Panxl and Panx3 are co-expressed in mouse epidermis and play distinct roles in regulating kératinocyte differentiation