Ferroptosis is a form of necrotic cell death caused by iron-dependent peroxidation of polyunsaturated phospholipids on cell membranes and is actively suppressed by the cellular antioxidant systems. We report here that oxidoreductases, including NADPH-cytochrome P450 reductase (POR) and NADH-cytochrome b5 reductase (CYB5R1), transfer electrons from NAD(P)H to oxygen to generate hydrogen peroxide, which subsequently reacts with iron to generate reactive hydroxyl radicals for the peroxidation of the polyunsaturated fatty acid (PUFA) chains of membrane phospholipids, thereby disrupting membrane integrity during ferroptosis. Genetic knockout of POR and CYB5R1 decreases cellular hydrogen peroxide generation, preventing lipid peroxidation and ferroptosis. Moreover, POR knockdown in mouse liver prevents ConA-induced liver damage. Ferroptosis, therefore, is a result of incidental electron transfer carried out by POR/CYB5R1 oxidoreductase and thus needs to be constitutively countered by the antioxidant systems. Display omitted •The oxidoreductases POR and CYB5R1 are required for ferroptosis•POR and CYB5R1 produce H2O2 to initiate iron-dependent Fenton reaction•POR and CYB5R1 induce lipid peroxidation and membrane rupture in liposomes•Knockdown of POR in vivo protects ConA-induced ferroptotic liver damage Yan et al. found that, during ferroptosis, the oxidoreductases POR and CYB5R1 transfer electrons from NAD(P)H to oxygen to generate H2O2, which reacts with iron to produce hydroxyl radials for peroxidation of PUFA-containing membrane phospholipids to cause membrane damage and necrotic cell death.