Objective To investigate the effects of Toll-like receptors (TLR3/TLR4/TLR5/TLR7) on the development, prognosis and immune characteristics of rectal cancer and to explore the association of genetic variation in the regulation region of TLRs with the risk for rectal cancer. Methods Gene expression profiling interactive analysis (GEPIA) platform was used to analyze the expression of TLRs in rectal cancer and its relationship with prognosis. Transcriptome data and clinical data of rectal cancer were downloaded from TCGA database and the correlation between TLRs expression and pathological stage was analyzed. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to determine the genotype of TLR4 rs1927914 and rs7869402 polymorphism. TaqMan probe method was used to determine the genotypes of TLR3 rs5743303, TLR5 rs1640816 and TLR7 rs7869402 variants. The association of TLRs genetic variants with the rectal cancer risk was analyzed by unconditional logistic regression. The immune cells in rectal cancer samples were scored using ESTIMATE algorithm. Using TIMER online data platform, the relationship between both the expression of TLRs and copy number variation (CNV) of genes and immune infiltrated cells was evaluated. Gene function related to TLR3 expression was evaluated by GO function enrichment analysis, gene set variation analysis(GSVA) was used to predict the regulatory network of TLR3 in rectal cancer. Results TLR3 has a lower expression in rectal cancer tissues than in adjacent tissues, which was related to poor prognosis. Compared with the individuals carrying TLR3 AA genotype, the individuals with at least one TLR3 rs5743303 T allele had significantly higher risk of rectal cancer (OR=1.43, 95%CI 1.07-1.91).The study did not show that TLR4 rs1927914, TLR4 rs7869402, TLR5 rs1640816 and TLR7 rs3853839 polymorphism had effect on the risk for rectal cancer. After stratified analysis, the data showed that TLR3 rs5743303 AT or TT contributed the susceptibility to rectal cancer in males (OR=1.47, 95%CI 1.01-2.11), younger subjects (OR=1.64, 95%CI 1.08-2.47), smokers (OR=2.42, 95%CI 1.37-4.38) and drinkers (OR=2.70, 95%CI 1.52-4.80). ESTIMATE and TIMER analyses showed that TLR3 effected on the immune cell infiltration. The results of GO functional enrichment analysis showed that TLR3 was mainly related to changes in related functions such as receptor ligand activity, antigen binding, immunoglobulin receptor binding, growth factors and cytokines. GSVA results showed that TLR3 inhibited the development of rectal cancer by regulating metabolic or immune-related pathways, such as cell apoptosis, natural killer cell-mediated cytotoxicity, phospholipid metabolism and autophagy regulation. Conclusion TLR3 effects on the occurrence and prognosis of rectal cancer and rs5743303 polymorphism increase the susceptibility to rectal cancer.