Esophageal cancer is one of the most aggressive cancers in the world, 70% of which are from China and esophageal squamous cell carcinoma (ESCC) is the major histopathological form (>90%). The single nucleotide polymorphisms (SNPs) in mature sequence of microRNA (miRNA) (mmSNPs) could cause the alteration of microRNA expression and contribute to the susceptibility of cancers. To evaluate the association between mmSNPs and ESCC, a case-control study including 773 patients with ESCC and 882 gender- and age-matched controls was carried out to investigate the association of five mmSNPs (miR-449b rs10061133, miR-4293 rs12220909, miR-608 rs4919510, miR-627 rs2620381, and miR-646 rs6513497) with ESCC susceptibility. As a result, two SNPs, miR-449b rs10061133 and miR-4293 rs12220909, were associated with decreased ESCC risk. For miR-449b rs10061133 A>G, individuals carrying GG genotype had an odds ratio (OR) of 0.77 (95% confidence interval (95% CI) 0.62-0.97) compared with individuals with AA genotype. In the recessive model, the GG genotype also showed a protective effect on ESCC (OR = 0.78, 95% CI 0.63-0.97). For miR-4293 rs12220909 G>C, the heterozygous genotype GC was associated with a decreased ESCC risk (OR = 0.77, 95% CI 0.61-0.97) compared with GG genotype. The C allele conferred 23% decrease in ESCC risk compared with the G allele in the allelic model (95% CI 0.63-0.93). In the dominant model, the GC/CC genotypes decreased the risk of ESCC (adjusted OR = 0.77, 95% CI 0.61-0.96). This study provides the first evidence that miR-449b rs10061133 and miR-4293 rs12220909 are associated with ESCC risk in Chinese population.