Objective To determine the expression and activation of nuclear factor kappa Bp65 (NF-κBp65) protein in human middle ear cholesteatoma epithelium, and to explore its possible role in the pathogenesis of middle ear cholesteatoma. Methods SP immunohistochemical assay was used for detecting the expression of NFκBp65 protein in 30 specimens of human middle ear cholesteatoma epithelium and 15 specimens of normal skin from the external ear canal. Results NF-κBp65 immunoreactivity was detected in the nuclei of epithelial cells. The positive expression rate of NF-κBp65 protein in middle ear cholesteatoma epithelium was 63.3% (19/30), while that in normal skin specimens was 20.0% (3/15). The difference between them was significant (P<0.01). Conclusion The abnormal expression of NF-κBp65 in middle ear cholesteatoma epithelium indicates its important role in the formation and development of cholesteatoma. The activation of NF-κBp65 might be an important factor involved in the hyperproliferation of cholesteatoma epithelium.