In this study, the association between the 729G/C polymorphism in Toll-like receptor 4 (TLR4) and the risk of bladder cancer was investigated. A total of 376 patients with bladder cancer and 380 healthy volunteers from the Third Xiangya Hospital of Central South University (China) were enrolled in this study between January 2008 and February 2014. The TLR4-729G/C polymorphism was detected by the polymerase chain reaction-restriction fragment length polymorphism assay. There was a significant difference in the distribution of the TLR4-729G/C genotype between bladder cancer patients and healthy controls (P < 0.001). Our analysis showed that the GC genotype (OR = 2.99; 95%CI = 1.01-4.81, P = 0.046) and CC genotype (OR = 3.67; 95%CI = 2.11-7.27, P = 0.017) were significantly associated with increased bladder cancer risk when the GG genotype served as a reference. Furthermore, carriers of the C allele had a significantly increased risk of developing bladder cancer (OR = 3.89; 95%CI = 2.88-8.53; P = 0.009). Our results suggest a correlation between the TLR4-729G/C polymorphism and the risk of developing bladder cancer in this Chinese population.