Nuclear factor-κB (NF-κB)/p65, a vital signaling molecule in the NF-κB pathway, participates in diverse physiological functions and host-virus interactions. However, the involvement of NF-κB/p65 in fish virus infection remains poorly understood. In this study, we explored the role of the p65 in virus infection and its impact on IL-18 regulation in largemouth bass (Micropterus salmoides). Bioinformatics analysis showed that the ORF sequence of Msp65 spanned 1941 bp, encoding 646 amino acids with two conserved functional domains, including RHD and IPT domain. Msp65 mRNA was presented in various tissues, with higher levels detected in the liver and gill. After exposure to largemouth bass virus (LMBV), red grouper nervous necrosis virus, lipopolysaccharide and poly (I:C), Msp65 expression was activated in vivo. In addition, the antiviral role of Msp65 were explored. In vitro, Msp65 overexpression hindered LMBV replication and formation of viral assembly site. In vivo, we found that disruption of Msp65 by using maslinic acid (MA) notably promoted the infectivity of LMBV, indicating its antiviral capabilities in largemouth bass. Besides, the downregulation of Msp65 suppressed the expression of inflammatory and interferon signaling molecules. Conversely, Msp65 overexpression boosted the activities of IFN-I, IFN-III and ISRE promoters, suggesting the positive regulation of Msp65 on interferon immune pathway. Furthermore, to unveil the regulatory role of Msp65 on MsIL-18, a promoter investigation was conducted. The luciferase reporter assay demonstrated that Msp65 positively influenced the expression of MsIL-18. Subsequent analysis suggested that the putative binding sites for MsIL-18 could potentially reside within the -228 to -203 bp of the MsIL-18 promoter. These findings illustrated that Msp65 involved in LMBV infection by modulating immune responses, presenting a novel insight into the antiviral mechanisms of p65 in bony fish.