Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive cancers in the world. Epidemiological survey studies have verified that the development of ESCC relates to a complex interactive process between multiple genetic susceptibilities and environmental exposure. Serpins are a broadly distributed family of protease inhibitors and have been recognized as tumor suppressors in multiple cancer types. While previous studies have reported that Serpin polymorphisms are associated with tumorigenesis, the genetic and functional single nucleotide polymorphisms (SNP) in these genes appear to be complex and remain to be elucidated. In this study, a total of 500 ESCC cases and 500 matched controls in a Southwest China population were evaluated for six SNPs in the exons of three Serpin genes (SerpinB5, SerpinB2, and SerpinE1). Among the six SNPs, the C allele of rs2289519 and rs2289520 in SerpinB5 showed decreased risk of ESCC and the variants might interact with smoking status. Haplotype analysis showed that the T-G haplotype (corresponding to rs2289519-rs2289520) increased the risk of ESCC, while the C-C haplotype decreased the risk. We also found that SerpinB5 gene mRNA expression was significantly downregulated in ESCC cell lines and patient specimen while there is no change in protein structure with different haplotypes. Our results demonstrated that the expression of SerpinB5 was downregulated in ESCC, and the positive SNPs might be associated with a risk of ESCC development.