We recently developed an original HCV-permissive cell culture model based on both partial differentiation and physiological oxygen pressure (Hypo-Diff cells) that produces highly infectious lipid-rich and native-like HCV-lipoviroparticles (LVPs). Here, we explored the precise role of physiological hypoxia and related specific transcription factors, hypoxia-inducible factors 1α and 2α (HIF-1α and HIF-2α) to better understand the mechanism governing viral morphogenesis. Knocking-down HIF-2α specifically reduced both the number and size of neutral lipid-rich droplets in Huh7.5 Hypo-Diff cells, suggesting a central role for HIF-2 in controlling lipid metabolism under physiological hypoxia. In HCV-infected siHIF-2α Hypo-Diff cells, both HCV replication and the specific infectious viral efficacy of progeny viruses were significantly impaired. Interestingly, the ectopic expression of a mutated form of the HIF-2α protein, stabilized in normoxia, both increased the number and size of neutral lipid-rich droplets and restored the production of highly infectious HCV viruses in the absence of cell differentiation. Finally, by iodixanol fractionation of supernatants to determine the mean buoyant density of infectious HCV LVPs, we established that HIF-2α is exclusively responsible for producing highly lipidated and broadly infectious HCV-LVPs by Hypo-Diff cells. These findings thus clearly establish the central role of physiological hypoxia, and notably HIF-2, in the production of highly infectious lipid-rich native-like HCV particles. Since physiological hypoxia is a shared characteristic in mammalian tissues, we propose to reconsider the role of natural oxygen tension and especially the role of HIF-2 in the life cycle of other lipid-associated viruses, whether hepatotropic or not.