The aims of this study were to investigate whether p53 gene codon 72 polymorphism was a biomarker associated with esophageal squamous cell carcinoma (ESCC) and its relationship with smoking status in China. The p53 genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism among 673 patients with ESCC and 694 healthy controls. The association between p53 genotypes and risk of developing ESCC was estimated by odds ratios (OR) and their 95% confidence intervals (CIs) computed by logistic regression. Compared with Arg/Arg homozygotes, Pro/Pro homozygotes had a nearly twofold increased risk (adjusted OR, 1.83; 95% CI, 1.35-2.48). For the Pro/Arg heterozygotes, there was no evident increased risk (adjusted OR, 1.01, 95% CI, 0.78-1.30). Furthermore, the risk associated with the Pro/Pro variant genotype was more pronounced in younger patients at diagnosis (= 45 years) (OR, 7.4; 95% CI, 1.44-37.89, P = 0.02), in women (OR, 3.15; 95% CI, 1.52-4.53, P = 0.02) and in non-smokers (OR, 2.49; 95% CI, 1.58-3.94) and light smokers (OR, 2.13; 95% CI, 1.15-3.93). But tests for homogeneity between smoking-related OR showed no significant differences (P = 0.4). The p53 gene codon 72 Pro/Pro genotype was significantly associated with the increased risk of ESCC in a Chinese mainland population and may be an independent factor in susceptibility to ESCC. The association was especially noteworthy in women and in younger patients.