Increased reports uncovered that mammalian tripartite motif-containing 62 (TRIM62) exerts crucial roles in cancer and innate immune response. However, the roles of fish TRIM62 in antiviral immune response remained uncertain. In this study, a TRIM62 gene was cloned from orange spotted grouper (EcTRIM62) and its roles in grouper RNA virus infection was elucidated in vitro. EcTRIM62 shared 99% and 83% identity to bicolor damselfish (Stegastes partitus) and human (Homo sapiens), respectively. Sequence alignment indicated that EcTRIM62 contained three domains, including a RING-finger domain, a B-box domain and a SPRY domain. In healthy grouper, the transcript of EcTRIM62 was predominantly detected in brain and liver, followed by heart, skin, spleen, fin, gill, intestine, and stomach. Subcellular localization analysis indicated that bright fluorescence spots were observed in the cytoplasm of EcTRIM62-transfected grouper spleen (GS) cells. During red-spotted grouper nervous necrosis (RGNNV) infection, overexpression of EcTRIM62 significantly enhanced the severity of CPE and increased viral gene transcriptions. Furthermore, the ectopic expression of EcTRIM62 significantly decreased the transcription level of interferon signaling molecules, including interferon regulatory factor 3 (IRF3), IRF7, interferon-stimulated gene 15 (ISG15), melanoma differentiation-associated protein 5 (MDA5), myxovirus resistance gene MXI, and MXII, suggesting that the negative regulation of interferon immune response by EcTRIM62 might directly contributed to its enhancing effect on RGNNV replication. Furthermore, our results also demonstrated that overexpression of EcTRIM62 was able to differently regulate the expression levels of pro-inflammation cytokines. In addition, we found the ectopic expression of EcTIRM62 negatively regulated MDA5-, but not mediator of IRF3 activation (MITA)-induced interferon immune response. Further studies showed that the deletion of RING domain and SPRY domain significantly affected the action of EcTRIM62, including the enhancing effect on virus replication and regulation of interferon immune response. Thus, our studies firstly demonstrated that EcTRIM62 negatively regulated the innate antiviral immune response against fish RNA viruses.