Abstract: In order to reveal the biological role of nonsynonymous SNP A1116T of porcine TLR3 Gene and to investigate the relationship of the SNP with disease resistance/susceptibility, the study was designed to construct eukaryotic expression vector of two types of alleles by splicing overlap extension PCR and site-directed mutation methods, to analyze the function of the two alleles in signal transduction in cultured PK-15 cell by dual-luciferase reporter assay and Real-time PCR methods. Two types of eukaryotic expression vectors of porcine TLR3 were successfully constructed. The dual-luciferase reporter assay demonstrated that the mutant type TLR3 allele was defective for activating TLR3-dependent reporter constructers. The real-time PCR analysis showed that the mutant type was reduced in inducing IL-6 transcription. The results indicate that the mutation influences the signal transduction ability of porcine TLR3.