The thymus plays an essential role in the maturing of progenitor cells to functional T cells. Recent studies suggest that the Hedgehog (Hh) signaling pathway contributes to this differentiation process. However, there is limited information concerning the expression of Hh pathway-related proteins (Hh proteins) in the human thymus. The staining of Hh proteins in the thymic epithelium of 26 surgically resected thymoma tissues was examined by immunohistochemistry. The staining of sonic Hh (Shh) correlated relatively well with the World Health Organization histological classification of thymoma. The higher the grade, the fainter the staining. However, no significant difference in Shh staining was found between normal and neoplastic epithelia. Interestingly, Gli1 staining in thymomas was significantly greater than that in normal thymus (p < 0.0001). Thus, some members of the Hh signaling pathway may contribute to the development of thymoma.