This study aims to scan the 5'-upstream regulatory region of the p21 gene to identify all putative functional single nucleotide polymorphisms (SNPs) and to evaluate the contribution of p21 variants to oesophageal squamous cell carcinoma (ESCC) in the Chinese Han population. Common SNPs were identified, and both locus-based and haplotype-based association tests were used to evaluate the potential risk of these p21 gene polymorphisms for ESCC. Immunohistochemistry assay was further performed to detect the P21 protein expression in ESCC specimens. Twenty three SNPs were identified and seven Tagging SNPs were chosen to represent all 23 SNPs. Univariate analysis indicated that the rs3829963 C and the rs2395655 G alleles increased susceptibility to ESCC (OR = 1.606 and OR = 1.572, respectively). The rs3829963 C and rs2395655 G alleles, combined with cigarette smoking, could further increase the risk for ESCC (OR = 2.657 and OR = 2.828, respectively). Additionally, the rs2395655 G allele appeared to elevate the positive rate of P21 expression in ESCC tissues, as compared to the A allele. This report demonstrates for the first time that rs3829963 and rs2395655, in the promoter of the p21 gene are potentially functional, modulating susceptibility to ESCC among the high-risk cigarette-smoking Chinese population.