Bladder cancer is the ninth most frequent malignancy in China. Toll-like receptor 4 (TLR4) is expressed on various cells and greatly involves in immune responses. Genetic polymorphism may affect the pathogenesis of diseases through various pathways. In the current study, we evaluated the association between genetic polymorphisms in TLR4 and risk of bladder cancer. We also examined the effect of the polymorphisms on gene expression. The TLR4 -729G/C and -260G/C polymorphisms were genotyped in 282 bladder cancer patients and 298 healthy controls in the Chinese population. Results showed that subjects with -729GC genotype are at significantly higher risk of bladder cancer than those with GG genotype [odds ratio (OR) = 2.50, 95% confidence interval (CI) = 1.39-4.48, P = 0.002]. Similarly, TLR4 -729C allele revealed a positive association with the disease (OR = 2.39, P = 0.002). The other polymorphism, TLR4 -260G/C, did not present clear correlations with bladder cancer. To understand the function of the polymorphisms, we evaluated TLR4 messenger RNA (mRNA) and protein levels in CD4+ T cells, CD8+ T cells, and monocytes from subjects carrying different TLR4 genotypes. Results revealed that subjects carrying -729GC genotype had significantly downregulated mRNA and protein levels of TLR4 in CD4+ T cells, CD8+ T cells, and monocytes compared to those carrying GG genotype. However, subjects with -260G/C polymorphism did not show any differences in gene expression from immune cells These data suggest that TLR4 polymorphism is associated with increased susceptibility to bladder cancer possibly by downregulating gene expression in various immune cells.