Esophageal squamous cell carcinoma (ESCC) is considered one of the most aggressive cancers with poor prognosis. The high molecular weight cytokeratin 34βE12 (CK34βE12) is recognized by the antibody, that is expressed in the cytoplasm of epithelial basal cells, and has been considered as a potential marker for prostate cancer, breast cancer, and basaloid carcinoma of the lung. However, there are no clinicopathological studies investigating CK34βE12 expression at the invasive front of ESCC. In this study, we examined 170 surgically resected cases of ESCC to clarify the clinicopathological significance of CK34βE12 expression. CK34βE12 expression was found in 85.3% (145/170) of ESCC cases and was significantly correlated with lymph node metastasis (66.2% [96/145], P = 0.034), depth of tumor invasion (57.9% [84/145], P = 0.042), and differentiation (82.1% [119/145], P = 0.013). These results indicated that CK34βE12 expression is a good indicator of lymph node metastasis, depth of tumor invasion, and differentiation in case of ESCC.