Dystrophin-associated glycoprotein 1 (DAG1) gene has been shown to act as a tumor suppressor gene in many human carcinomas. The association between polymorphism 2650C/T in DAG1 gene and Hepatitis B virus (HBV)-related Hepatocellular carcinoma (HCC) remains unclear. To examine this association, genotypes at the polymorphism locus 2650C/T (dbSNP: rs1801143) in the Hubei Han Chinese population were examined. 2051 subjects (468 HBV-related HCC patients, 1006 non-HCC patients with HBV infection and 577 healthy controls) were genotyped for the 2650C/T variant by TaqMan real time-polymerase chain reaction (PCR). Statistical analysis found a polymorphism 2650C/T-dependent HBV-related HCC susceptibility. HBV-related HCC patients showed significant differences in allelic distributions (P value <0.05) and genotypic frequencies (P value <0.05) when compared to both non-HCC patients with HBV infection (odds ratio [OR] = 1.758, 95% confidence interval [95% CI] = 1.672 similar to 1.848) and healthy controls (OR = 1.594, 95% CI = 1.046 similar to 2.428). Our research indicated that in the Hubei Han Chinese population, subjects with the T allele of rs1801143 show a higher susceptibility to HBV-related HCC compared to those with the C allele. To the best of our knowledge, this is the first study to investigate the association between polymorphism 2650C/T in the DAG1 gene and HBV-related HCC, which has promising application in early diagnosis and optimal therapy of HCC.