Purpose: p27kip1 belongs to the KIP/CIP family of cyclin-dependent kinase inhibitors and is considered to be a tumor suppressor. Involucrin has been known as a marker of differentiation of squamous cell carcinoma (SCC). The aim of this study was to evaluate the clinicopathologic significance of the expression of p27 and involucrin in esophageal SCC. Methods: Immunohistochemical expression of p27 and involucrin was examined in 70 specimens of esophageal SCC. The correlation of the expression of these proteins and clinicopathologic features was evaluated. Results: Cellular differentiation in esophageal SCC was significantly correlated with the expression of p27 and involucrin (p = 0.010 and p = 0.002, respectively). Among well, moderately and poorly differentiated SCCs, 45.8 ± 21.6, 20.0 ± 15.0 and 10.6 ± 9.1% of carcinoma cells expressed involucrin, respectively (p < 0.0001 for well vs. poorly, p < 0.0001 for well vs. moderately, and p = 0.042 for moderately vs. poorly). There existed a more powerful statistical difference regarding the histological grade between SCCs with the expression of both p27 and involucrin and tumors with other expression patterns (p = 0.0001). Conclusions: Expression of both p27 and involucrin can be a powerful biological marker of cellular differentiation of esophageal SCC.