Microglia are CNS resident innate immune cells of myeloid origin that become activated and produce innate proinflammatory molecules upon encountering bacteria or viruses. TLRs are a phylogenetically conserved diverse family of sensors for pathogen-associated molecular patterns that drive innate immune responses. We have recently shown that mice deficient in TLR3 (TLR3−/− mice) are resistant to lethal encephalitis and have reduced microglial activation after infection with West Nile virus, a retrovirus that produces dsRNA. We wished to determine whether microglia recognize dsRNA through the TLR3 pathway. In vitro, murine wild-type primary cultured microglia responded to synthetic dsRNA polyinosinic-polycytidylic acid (poly (I: C)) by increasing TLR3 and IFN-β mRNA and by morphologic activation. Furthermore, wild-type microglia dose dependently secreted TNF-α and IL-6 after poly (I: C) challenge, whereas TLR3−/− microglia produced diminished cytokines. Activation of MAPK occurred in a time-dependent fashion following poly (I: C) treatment of wild-type microglia, but happened with delayed kinetics in TLR3−/− microglia. As an in vivo model of encephalitis, wild-type or TLR3−/− mice were injected intracerebroventricularly with poly (I: C) or LPS, and microglial activation was assessed by cell surface marker or phospho-MAPK immunofluorescence. After intracerebroventricular injection of poly (I: C), microgliosis was clearly evident in wild-type mice but was nearly absent in TLR3−/− animals. When taken together, our results demonstrate that microglia recognize dsRNA through TLR3 and associated signaling molecules and suggest that these cells are key sensors of dsRNA-producing viruses that may invade the CNS.