Purpose: The present study was designed to elucidate the immunoreactivity and protein level of IL-17 in human cholesteatomas. Procedures: The expression and localization of IL-17 and receptor activator of nuclear factor ĸB ligand (RANKL) were examined by immunohistochemistry in tissue specimens collected from 24 patients with cholesteatomas. The cellular sources of IL-17 were assessed by double staining with CD4. The level of IL-17 protein was determined using an enzyme-linked immunosorbent assay. The degree of bone destruction was compared with the IL-17 immunoreactivity. Results: IL-17 immunoreactivity was detected in the inflammatory cells in the granulation tissues and there were increased levels of IL-17 protein. The localization of IL-17 expression coincided with CD4-positive lymphocytes. IL-17 was correlated with the cells positive for RANKL. The degree of bone destruction was dependent on the number of infiltrated IL-17-positive cells. IL-17-driven pathology was observed in the congenital type as compared with the acquired type. Conclusions: The present study suggests that IL-17 is related to the pathogenesis of the disease.