Abstract: Von Hippel-Landau tumor suppressor protein (VHL) is an E3 ubiquitin ligase that functions as a critical regulator of the oxygen-sensing pathway for targeting hypoxia-inducible factors. Recent evidence has shown that mammalian VHL may play important roles in NF-κB signaling pathways, but the molecular regulatory mechanisms remain unclear. Herein, the roles of mandarin fish (<i>Siniperca chuatsi</i>) VHL (<i>sc</i>VHL) in the NF-κB signaling pathways and mandarin fish ranavirus (MRV) replication were employed. The transcription of <i>sc</i>VHL occurred in response to immune stimulation and MRV infection, indicating that <i>sc</i>VHL might play an important role in innate immunity. Dual-luciferase reporter gene assays and RT‒qPCR results showed that <i>sc</i>VHL could evoke and positively regulate the NF-κB signaling pathways. The NF-κB signaling pathway inhibitor treatment results showed that the role of <i>sc</i>VHL in NF-κB signaling might be targeted to <i>sc</i>IKKα, <i>sc</i>IKKβ, <i>sc</i>IκBα or <i>sc</i>p65. Co-IP analysis results showed that <i>sc</i>IκBα was a novel target protein of <i>sc</i>VHL. Moreover, <i>sc</i>VHL targeted <i>sc</i>IκBα to catalyze the formation of K63-linked polyubiquitin chains to activate NF-κB signaling pathways. After MRV infection, the NF-κB signaling pathway remained activated, and the activation of NF-κB could promote MRV replication. These observations suggested that <i>sc</i>VHL could positively regulate NF-κB and significantly enhance MRV replication. Our study demonstrates a novel role of <i>sc</i>VHL in the NF-κB signaling pathway and fish virus infection.