The von Hippel-Lindau tumor suppressor protein (VHL), an E3 ubiquitin ligase, functions as a critical regulator of the oxygen-sensing pathway for targeting hypoxia-inducible factors. Recent evidence suggests that mammalian VHL may also be critical to the NF-κB signaling pathway, although the specific molecular mechanisms remain unclear. Herein, the roles of mandarin fish ( ) VHL ( VHL) in the NF-κB signaling pathway and mandarin fish ranavirus (MRV) replication were explored. The transcription of VHL was induced by immune stimulation and MRV infection, indicating a potential role in innate immunity. Dual-luciferase reporter gene assays and reverse transcription quantitative PCR (RT-qPCR) results demonstrated that VHL evoked and positively regulated the NF-κB signaling pathway. Treatment with NF-κB signaling pathway inhibitors indicated that the role of VHL may be mediated through IKKα, IKKβ, IκBα, or p65. Co-immunoprecipitation (Co-IP) analysis identified IκBα as a novel target protein of VHL. Moreover, VHL targeted IκBα to catalyze the formation of K63-linked polyubiquitin chains to activate the NF-κB signaling pathway. Following MRV infection, NF-κB signaling remained activated, which, in turn, promoted MRV replication. These findings suggest that VHL not only positively regulates NF-κB but also significantly enhances MRV replication. This study reveals a novel function of VHL in NF-κB signaling and viral infection in fish.