Innate immunity is required for effective control of poxvirus infections, but cellular receptors that initiate the host response to these DNA viruses remain poorly defined. Given this information and the fact that functions of TLRs in immunity to DNA viruses remain controversial, we investigated effects of TLR3 on pathogenesis of vaccinia virus, a prototype poxvirus. We used a recombinant strain Western Reserve vaccinia virus that expresses firefly luciferase to infect wild-type C57BL/6 and TLR3-/- mice through intranasal inoculation. Bioluminescence imaging showed that TLR3-/- mice had substantially lower viral replication in the respiratory tract and diminished dissemination of virus to abdominal organs. Mice lacking TLR3 had reduced disease morbidity, as measured by decreased weight loss and hypothermia after infection. Importantly, TLR3-/- mice also had improved survival relative to wild-type mice. Infected TLR3-/- mice had significantly reduced lung inflammation and recruitment of leukocytes to the lung. Mice lacking TLR3 also had lower levels of inflammatory cytokines, including IL-6, MCP-1, and TNF-alpha in serum and/or bronchoalveolar lavage fluid, but levels of IFN-beta did not differ between genotypes of mice. To our knowledge, our findings show for the first time that interactions between TLR3 and vaccinia increase viral replication and contribute to detrimental effects of the host immune response to poxviruses.