Innate immunity provides the first line of host defense against invading microbial pathogens. This defense involves retinoic acid-inducible gene-I-like receptors that detect viral RNA and activate the mitochondrial antiviral-signaling (MAVS) protein, an adaptor protein, leading to activation of the innate antiviral immune response. The mechanisms by which the MAVS signalosome assembles on mitochondria are only partially understood. Here, we identify tripartite motif 14 (TRIM14) as a mediator in the immune response against viral infection. TRIM14 localizes to the outer membrane of mitochondria and interacts with MAVS. Upon viral infection, TRIM14 undergoes Lys-63-linked polyubiquitination at Lys-365 and recruits NF-κB essential modulator to the MAVS signalosome, leading to the activation of both the IFN regulatory factor 3 and NF-κB pathways. Knockdown of TRIM14 disrupts the association between NF-κB essential modulator and MAVS and attenuates the antiviral response. Our results indicate that TRIM14 is a component of the mitochondrial antiviral immunity that facilitates the immune response mediated by retinoic acid-inducible gene-I-like receptors.