Toll-like receptors (TLR) play an important role in innate immunity to virus infections. We investigated the role of TLR3 in the pathogenesis of H5N1 and pandemic H1N1 (pH1N1) influenza virus infection in mice. The results of TLR3 was compared to that of wild type (WT) mice. WT mice and those defective in TLR3 were infected with influenza A/HK/486/97 (H5N1) or A/HK/415742/09 pH1N1 virus. As a comparison, mice with a defective MyD88 gene were also infected with the viruses. Survival and body weight loss were monitored for 14 days, and lung pathology, immune cells profile, viral load and cytokine responses were studied. Compared to wild-type mice, TLR3-/- mice have a survival advantage, a significantly faster regain of body weight, lower lung viral titer and less lung pathology following challenge with H5N1 virus. Such a survival advantage is not seen in pH1N1 infected TLR3-/- mice. On the contrary, MyD88-/- mice have an increased viral titer and decreased leukocyte infiltration in the lungs after infection with H5N1 virus and a poorer survival in pH1N1 infection. In conclusion, TLR3 has a detrimental effect on the pathogenesis of H5N1 infection but not in pH1N1 infection highlighting the differences in the pathogenesis of these two viruses and in the role of TLR3 in such pathogenesis.