The purpose of this study was to analyze loss of p16 expression and its relationship to hypermethylation, clinicopathological parameters and prognosis in patients with esophageal squamous cell carcinoma (ESCC). Tissue samples from 60 ESCC were subjected to histological analysis. Immunohistochemical staining for p16 expression was performed. DNA was extracted from these primary esophageal tumors and from sera from another 38 ESCC patients. The DNA was modified with bisulfite and analyzed for p16 promoter methylation by methylation-specific polymerase chain reaction. Twelve out of the 60 tumors (20%) were methylated at the p16 promoter and 48 tumors (80%) were unmethylated. There were no significant correlations between the methylation of the p16 promoter and clinicopathological parameters. Immunohistochemical staining revealed that 41 of the 60 tumors (68.3%) were p16-negative and 19 tumors (31.7%) were p16-positive. The correlation between negative p16 immunohistochemical staining and methylation was statistically significant (P = 0.0084). No instances of p16 methylation and p16 positive immunostaining were found. There was a close correlation between loss of p16 expression and poorer prognosis in ESCC (P = 0.0517 in overall survival, P = 0.0478 in disease-free survival). The p16 gene promoter hypermethylation was detected in the serum of two of 38 (5.2%) patients with ESCC. This indicates that p16 promoter methylation suppresses p16 expression and that the loss of expression has a close relationship with poor prognosis in patients with ESCC. The present results may lead to the development of new therapeutic strategies, such as p16(INK4A) gene therapy, to treat patients with ESCC.