Background & Aims: Hepatitis C virus (HCV) co-opts the very-low-density lipoprotein pathway for morphogenesis, maturation, and secretion, and circulates as lipoviroparticles (LVPs). We investigated the functions and underlying mechanisms of the lipid-associated TM6SF2 protein in modulating LVP formation and the HCV life cycle. Methods: We knocked down or overexpressed TM6SF2 in hepatic cells and examined HCV infection, measuring viral RNA and protein levels and infectious LVP titers. The density of secreted LVPs was evaluated by iodixanol gradient assay. We measured levels and patterns of TM6SF2 in liver biopsies from 73 patients with chronic hepatitis C, livers of HCV-infected humanized Alb-uPA/SCID/beige mice, and HCV-infected Huh7.5.1 cells. Results: TM6SF2 knockdown in hepatocytes reduced viral RNA and infectious viral particle secretion without affecting HCV genome replication, translation, or assembly. Overexpression of TM6SF2 reduced intracellular levels of HCV RNA and infectious LVPs, and conversely increased their levels in the culture supernatants. In HCV-infected cells, TM6SF2 overexpression resulted in production of more infectious LVPs in the lower-density fractions of supernatant. HCV infection increased TM6SF2 expression in cultured cells, humanized livers of mice, and liver tissues of HCV patients. TM6SF2 messenger RNA levels correlated positively with HCV RNA levels in liver biopsies from patients. SREBF2 appears to mediate the ability of HCV to increase the expression of TM6SF2 in hepatic cells. Conclusions: In studies of cells, mice and human liver tissues, we found TM6SF2 is required for maturation, lipidation, and secretion of infectious LVPs. HCV, in turn, up-regulates expression of TM6SF2 to facilitate productive infection.