Mitochondrial antiviral signaling protein (MAVS), also known as IPS-1, VISA, and Cardif, has been well studied for its crucial roles in the mammalian interferon immune response. To better understand the actions of MAVS in fish immune response, a MAVS homolog from orange spotted grouper (Epinephelus coioides) (EcMAVS) was cloned and characterized in this study. EcMAVS encoded a 563-amino acid peptide which showed 64% and 20% identity to rock bream (Oplegnathus fasciatus) and human (Homo sapiens), respectively. Sequence alignment analysis showed that EcMAVS shared a conserved CARD domain at N terminal, a central proline-rich region and a TM domain at C terminal. Phylogenetic analysis indicated that EcMAVS showed the nearest relationship to rock bream, followed by other fishes, birds and mammals. In healthy grouper, the transcript of EcMAVS was predominantly detected in gill, intestine and skin. In vitro, the expression level of EcMAVS was significantly increased during red-spotted grouper nervous necrosis virus (RGNNV) infection, but only slightly increased at the late stage of Singapore grouper iridovirus (SGIV) infection, suggested the EcMAVS might exert various roles in response to different viruses. Subcellular localization analysis showed that the fluorescence in EcMAVS transfected cells primarily co-localized with mitochondria. Overexpression of EcMAVS in grouper cells significantly inhibited the replication of RGNNV, demonstrated by the delay of CPE progression and the decrease of viral gene transcription. Differently, the replication of SGIV was almost not affected by the ectopic expression of EcMAVS. Furthermore, our results also showed that EcMAVS overexpression significantly increased the expression of interferon related cytokines, and activated both IRF3- and IRF7-mediated interferon promoter activities. Taken together, our results demonstrated that grouper MAVS exerted antiviral function against nodavirus infection via up-regulating the interferon immune response.