Nicotinic acetylcholine receptors are important regulators of smoking behavior and tobacco carcinogenesis. We studied the association of the CHRNB3-A6 variant rs13280604 in relation to esophageal squamous cell carcinoma (ESCC) in Chinese populations. Two independent case-control studies were conducted. The first case-control study, consisted of 866 ESCC patients and 1621 healthy controls from Northern China, and the second case-control study consisted of 853 ESCC patients and 860 unrelated controls from Southern China. A logistic regression model was used to evaluate the associations of rs13280604 with cancer risk. We found that Rs13280604 GG/AG genotypes were significantly associated with increased risk for ESCC in both case-control studies from Northern [odds ratio (OR), 1.42, 95% confidence interval (CI), 1.19-1.70, P = 1.1×10(-4)], Southern China (OR, 1.56, 95% CI, 1.26-1.93, P = 5.2×10(-5)), and the combined population of both studies (OR, 1.44, 95% CI, 1.26-1.65, P = 8.7×10(-8)), respectively. Our results suggest that this CHRNB3-A6 variant confers susceptibility to ESCC risk. However, future larger studies are needed to validate our finding.