Chromosome 1 open reading frame 10 (C1orf10) is either down-regulated or absent in esophageal squamous cell carcinoma (ESCC) tissues compared to its corresponding normal counterparts, and it is involved in heat shock and ethanol response and is expected to protect esophageal epithelium from damage. In the present study, we sequenced DNA samples from 32 individuals to search for genetic variants in the promoter region, coding region, and the untranslated region of C1orf10. Genotypes were analyzed in 991 patients and 984 controls. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated by logistic regression. Luciferase assays were carried out to find the functional SNPs. Six strongly linked single nucleotide polymorphisms (SNPs) spanning a region of 7 kb, -1747G/T, -1139G/C, -1079G/A, -900G/T, Gly480Ser, and 4666G/A were identified (D'= 1, r(2 )= 1). Only one SNP -1139G/C was selected to analyze the association between C1orf10 genotypes and risk of ESCC. Subjects with the -1139CC genotype had a greater risk of developing ESCC compared with those with the -1139GG genotype (adjusted OR = 1.34; 95% CI, 1.02-1.76). There appears to be an interaction between the -1139G/C polymorphism and tobacco smoking that contributes to the risk for ESCC. However, we did not detect any obvious difference in reporter gene assay driven by each allele of C1orf10 promoter or 3' UTR. These data showed that C1orf10 haplotypes containing -1747G/T, -1139G/C, -1079G/A, -900G/T, Gly480Ser, and 4666G/A are significantly associated with susceptibility to ESCC.