Ferroptosis, a form of regulated cell death induced by excessive lipid peroxidation, has recently emerged as a key tumor suppression mechanism(1–4). Glutathione peroxidase 4 (GPX4)(5, 6) and ferroptosis suppressor protein 1 (FSP1)(7, 8) constitute two major ferroptosis defense systems. Here we show that treatment with GPX4 inhibitors in cancer cells results in an acute depletion of N-carbamoyl-L-aspartate (C-Asp), a pyrimidine biosynthesis intermediate, with a concomitant accumulation of uridine. Supplementation of dihydroorotate (DHO) or orotate (OA), the substrate and product of DHO dehydrogenase (DHODH), attenuates or potentiates ferroptosis induced by GPX4 inhibition, respectively, and these effects are particularly pronounced in cancer cells with low expression of GPX4 (GPX4(low)). DHODH inactivation induces extensive mitochondrial lipid peroxidation and ferroptosis in GPX4(low) cancer cells, whereas DHODH inactivation synergizes with ferroptosis inducers to induce mitochondrial lipid peroxidation and ferroptosis in GPX4(high) cancer cells. Mechanistically, DHODH operates in parallel to mitochondrial GPX4 (but independent of cytosolic GPX4 or FSP1) to inhibit ferroptosis in the mitochondrial inner membrane, through reducing ubiquinone (CoQ) to ubiquinol (CoQH(2)), a radical-trapping antioxidant with anti-ferroptosis activity. We further show that the DHODH inhibitor brequinar selectively suppresses GPX4(low) tumor growth through inducing ferroptosis, whereas combined treatment with brequinar and sulfasalazine, an FDA-approved drug with ferroptosis inducing activity, synergistically induces ferroptosis and suppresses GPX4(high) tumor growth. Our results identify a DHODH-mediated ferroptosis defense mechanism in mitochondria and suggest a therapeutic strategy to target ferroptosis in cancer treatment.