Cholesterol sulfate is abundant in the human epidermis and is a putative natural ligand for retinoic acid receptor-related orphan receptor alpha (RORα). Although direct binding of cholesterol sulfate is expected to activate RORα, cholesterol sulfate can also induce RORα expression and increase RORα target gene expression. The purpose of this study was to determine whether cholesterol sulfate induces profilaggrin expression, a precursor of the barrier protein filaggrin in the epidermis, through activation of RORα by directly binding to RORα, or through increased RORα expression. Immunohistochemical and polymerase chain reaction (PCR) analyses showed that RORα was expressed in normal human epidermal keratinocytes (NHEKs) and that its expression increased during keratinocyte differentiation in parallel with that of profilaggrin and cholesterol sulfotransferase, which catalyzes the synthesis of cholesterol sulfate. Exogenous cholesterol sulfate significantly increased both RORα and profilaggrin expression in NHEKs, whereas no effect on profilaggrin expression was observed in cells in which RORα was knocked down with small interfering RNA (siRNA). Additionally, a luciferase reporter gene assay revealed that exogenous RORα dose-dependently increased the activity of the profilaggrin gene promoter even in the absence of cholesterol sulfate, and that this response involves activator protein-1. In conclusion, the results of this study indicate that cholesterol sulfate induces filaggrin expression through increased RORα expression. Further studies are required to fully elucidate the mechanisms involved.