Ferroptosis, a form of regulated cell death, is emerging as a key mechanism in tumor suppression. Glutathione peroxidase 4 (GPX4) and ferroptosis suppressor protein 1 (FSP1) are two major defense systems against ferroptosis. This study reveals that GPX4 inhibitors deplete N-carbamoyl-L-aspartate (C-Asp), a pyrimidine biosynthesis intermediate, leading to increased uridine levels. Supplementation with dihydroorotate (DHO) or orotate (OA) can either attenuate or potentiate ferroptosis, depending on the cancer cell's GPX4 expression level. DHODH, an enzyme localized in the mitochondrial inner membrane, operates in parallel with mitochondrial GPX4 to inhibit ferroptosis by reducing ubiquinone (CoQ) to ubiquinol (CoQH2), an antioxidant. Inactivation of DHODH induces extensive mitochondrial lipid peroxidation and ferroptosis in GPX4low cancer cells, while it synergizes with ferroptosis inducers to induce ferroptosis in GPX4high cancer cells. The DHODH inhibitor brequinar selectively suppresses GPX4low tumor growth through ferroptosis, and combined treatment with brequinar and sulfasalazine, an FDA-approved ferroptosis inducer, synergistically suppresses GPX4high tumor growth. These findings suggest that targeting DHODH-mediated ferroptosis defense could be a therapeutic strategy for cancer treatment.Ferroptosis, a form of regulated cell death, is emerging as a key mechanism in tumor suppression. Glutathione peroxidase 4 (GPX4) and ferroptosis suppressor protein 1 (FSP1) are two major defense systems against ferroptosis. This study reveals that GPX4 inhibitors deplete N-carbamoyl-L-aspartate (C-Asp), a pyrimidine biosynthesis intermediate, leading to increased uridine levels. Supplementation with dihydroorotate (DHO) or orotate (OA) can either attenuate or potentiate ferroptosis, depending on the cancer cell's GPX4 expression level. DHODH, an enzyme localized in the mitochondrial inner membrane, operates in parallel with mitochondrial GPX4 to inhibit ferroptosis by reducing ubiquinone (CoQ) to ubiquinol (CoQH2), an antioxidant. Inactivation of DHODH induces extensive mitochondrial lipid peroxidation and ferroptosis in GPX4low cancer cells, while it synergizes with ferroptosis inducers to induce ferroptosis in GPX4high cancer cells. The DHODH inhibitor brequinar selectively suppresses GPX4low tumor growth through ferroptosis, and combined treatment with brequinar and sulfasalazine, an FDA-approved ferroptosis inducer, synergistically suppresses GPX4high tumor growth. These findings suggest that targeting DHODH-mediated ferroptosis defense could be a therapeutic strategy for cancer treatment.