Viral infection triggers host innate immune responses through activation of the transcription factors NF-κB and IRF3, which coordinately regulate the expression of type-I interferons such as interferon-β (IFN-β). Herein, we report the identification of a novel protein termed MAVS (mitochondrial antiviral signaling), which mediates the activation of NF-κB and IRF3 in response to viral infection. Silencing of MAVS expression through RNA interference abolishes the activation of NF-κB and IRF3 by viruses, thereby permitting viral replication. Conversely, overexpression of MAVS induces the expression of IFN-β through activation of NF-κB and IRF3, thus boosting antiviral immunity. Epistasis experiments show that MAVS is required for the phosphorylation of IRF3 and IκB and functions downstream of RIG-I, an intracellular receptor for viral RNA. MAVS contains an N-terminal CARD-like domain and a C-terminal transmembrane domain, both of which are essential for MAVS signaling. The transmembrane domain targets MAVS to the mitochondria, implicating a new role of mitochondria in innate immunity.