The metabolic oxidative degradation of cellular lipids severely restricts replication of hepatitis C virus (HCV), a leading cause of chronic liver disease, but little is known about the factors regulating this process in infected cells. Here we show that HCV is restricted by an iron-dependent mechanism resembling the one triggering ferroptosis, an iron-dependent form of non-apoptotic cell death, and mediated by the non-canonical desaturation of oleate to Mead acid and other highly unsaturated fatty acids by fatty acid desaturase 2 (FADS2). Genetic depletion and ectopic expression experiments show FADS2 is a key determinant of cellular sensitivity to ferroptosis. Inhibiting FADS2 markedly enhances HCV replication, whereas the ferroptosis-inducing compound erastin alters conformation of the HCV replicase and sensitizes it to direct-acting antiviral agents targeting the viral protease. Our results identify FADS2 as a rate-limiting factor in ferroptosis, and suggest the possibility of pharmacologically manipulating the ferroptosis pathway to attenuate viral replication.