Hepatitis B virus (HBV) infection is one of the major causes of chronic liver inflammation. Toll-like receptor 3 (TLR3) plays a key role in innate immunity and is responsible for recognizing viral pathogens. It has been reported that the TLR3 C1234T polymorphism is associated with various diseases. The aim of this study was to investigate whether TLR3 polymorphisms were correlated with susceptibility to chronic HBV infection. Two polymorphisms in the TLR3 gene, A952T and C1234T, were tested by direct sequencing in 452 chronic hepatitis B (CHB) patients and 462 healthy controls. Data showed that subjects carrying 1234CT genotype and TT genotype had 1.42-fold and 2.31-fold increased risk of chronic HBV infection compared to those with CC genotype (95 % confidence interval [CI] = 1.08-1.86, p = 0.012; 95 % CI = 1.34-3.96, p = 0.002, respectively). Further analysis revealed that the prevalence of 1234CT genotype and T allele was significantly increased in CHB patients with acute-on-chronic liver failure (ACLF) than those without ACLF (odds ratio [OR] = 1.55, p = 0.030; OR = 1.43, p = 0.040, respectively). These results indicate that TLR3 C1234T polymorphism could be a risk factor for the development of chronic HBV infection, especially the CHB-related ACLF.