The "cytokine storm" and excessive inflammation triggered by lethal avian influenza virus (IAV) are responsible for its high virulence and mortality. However, the molecular mechanism behind these effects is unclear. In this study, we used LA795 cells and a mouse model to assess the crucial role of TLR3 during infection with lethal avian influenza A virus and subsequent inflammation. The results showed that IAVs could replicate and proliferate well in LA795 cells and that the replication of H5N1 was more e?cient than human H1N1 and lowly pathogenic avian H7N2 viruses. The TLR3 signaling pathways were activated preferentially in vitro and in vivo and a range of pro-inflammatory cytokines were released following H5N1 infection. RNAi and TLR3 knockout mice were used to validate the results. These results are the first to provide insight into the preferential involvement of TLR3 in lethal avian influenza A virus infection and inflammation compared with others such as human or lowly pathogenic avian influenza A viruses. The data will increase understanding of the pathogenesis of lethal avian influenza A virus infection and may help facilitate the development of novel therapeutic aids targeting TLR3 signaling pathways.