Activation of the Hedgehog (HH) signaling pathway plays a critical role in the development of basal cell carcinoma (BCC). HH signaling activity is produced by nuclear translocation of transcription factors, glioma-associated oncogene homolog (GLI). Among three GLI subfamilies, GLI1 is the only full-length transcriptional activator, and its nuclear localization is recognized as a signature event in HH signaling activation. However, limited published work has investigated the nuclear staining of GLI1 protein in human tumor tissue samples by immunohistochemical analysis. In this study, we performed immunohistochemical staining of GLI1 in 382 cases of cutaneous epithelial tumors, including 196 BCC cases, using rabbit monoclonal antihuman GLI1 antibody (C68H3). As a result, 98.2% cases of BCC showed a diffuse and strong nuclear staining pattern regardless of the histological subtype. Positive staining was mainly restricted to the tumor nests, while the overlying epidermis was negative suggesting specificity of the antibody. In further analysis of other cutaneous epithelial tumors, 100% (4/4) cases of trichoblastoma, 15.1% (5/33) Bowen's disease, 3.5% (1/28) actinic keratosis and 12.5% (4/32) squamous cell carcinoma showed the nuclear staining pattern of GLI1. This suggested that HH signaling is also dysregulated in some other cutaneous malignant tumors. In conclusion, the C68H3 antibody is a useful tool for revealing activation of HH signaling in immunohistochemical analysis.