Melanoma differentiation-associated gene 5 (MDA5) is a critical member of retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) family which can recognize viral RNA and enhances antiviral response in host cells. In this study, a MDA5 homolog from orange spotted grouper (Epinephelus coioides) (EcMDA5) was cloned, and its roles on grouper virus infection were characterized. The full-length EcMDA5 cDNA encoded a polypeptide of 982 amino acids with 74% identity with MDA5 homolog from rock bream (Oplegnathus fasciatus). Amino acid alignment analysis indicated that EcMDA5 contained three functional domains: two caspase activation and recruitment domain (CARDs), a DEAD box helicase-like (DExDc) domain, a helicase superfamily C-terminal domain (HELICc), and a C-terminal regulatory domain (RD). Upon challenge with Singapore grouper iridovirus (SGIV) or polyinosin-polycytidylic acid (poly I:C), the transcript of EcMDA5 was significantly up-regulated especially at the early stage post-injection. Under fluorescence microscopy, we observed that EcMDA5 mostly localized in the cytoplasm of grouper spleen (GS) cells. Interestingly, during virus infection, the distribution pattern of EcMDA5 was significantly altered in SGIV infected cells, but not in red spotted grouper nervous necrosis virus (RGNNV) infected cells, suggested that EcMDA5 might interact with viral proteins during SGIV infection. The ectopic expression of EcMDA5 in vitro obviously delayed virus infection induced cytopathic effect (CPE) progression and significantly inhibited viral gene transcription of RGNNV and SGIV. Moreover, overexpression of EcMDA5 not only significantly increased interferon (IFN) and IFN-stimulated response element (ISRE) promoter activities in a dose dependent manner, but also enhanced the expression of IRF3, IRF7 and TRAF6. In addition, the transcription level of the proinflammatory factors, including TNF-α, IL-6 and IL-8 were differently altered by EcMDA5 overexpression during SGIV or RGNNV infection, suggesting that the regulation on proinflammatory cytokines by EcMDA5 were also important for RGNNV infection. Together, our results demonstrated for the first time that the inhibitory effect of fish MDA5 on iridovirus replication might be mainly through the regulation of proinflammatory cytokines.