This study identifies a novel therapeutic target for cancer treatment by demonstrating that DHODH (dihydroorotate dehydrogenase) plays a critical role in mitochondrial ferroptosis defense. Ferroptosis is a form of regulated cell death driven by lipid peroxidation, and it has emerged as a key tumor suppression mechanism. The study shows that GPX4 (glutathione peroxidase 4) and FSP1 (ferroptosis suppressor protein 1) are two major ferroptosis defense systems. However, the study reveals that DHODH operates in parallel to mitochondrial GPX4 to inhibit ferroptosis by reducing ubiquinone (CoQ) to ubiquinol (CoQH₂), a radical-trapping antioxidant with anti-ferroptosis activity. DHODH inhibition leads to mitochondrial lipid peroxidation and ferroptosis, particularly in cancer cells with low GPX4 expression. Conversely, DHODH activation can protect cells from ferroptosis. The study further shows that DHODH inhibitors, such as brequinar, selectively suppress GPX4-low tumor growth by inducing ferroptosis. When combined with ferroptosis inducers like sulfasalazine, DHODH inhibitors synergistically induce ferroptosis and suppress GPX4-high tumor growth. These findings suggest that targeting DHODH could be a promising strategy for cancer therapy, especially in GPX4-low cancers. The study also highlights the importance of DHODH in mitochondrial lipid peroxidation and ferroptosis. DHODH is localized on the outer face of the mitochondrial inner membrane and couples the oxidation of DHO to OA with the reduction of CoQ to CoQH₂. This process is essential for suppressing ferroptosis. The study further demonstrates that DHODH inhibition can sensitize GPX4-high cancer cells to ferroptosis, while DHODH deletion induces ferroptosis in GPX4-low cancer cells. These findings suggest that DHODH is a key regulator of mitochondrial ferroptosis and that targeting DHODH could be a viable therapeutic approach for cancer treatment. The study also shows that DHODH inhibitors, such as brequinar, have potential as cancer therapeutics, especially when combined with other ferroptosis-inducing agents like sulfasalazine. Overall, the study provides new insights into the role of DHODH in ferroptosis and highlights its potential as a therapeutic target in cancer treatment.This study identifies a novel therapeutic target for cancer treatment by demonstrating that DHODH (dihydroorotate dehydrogenase) plays a critical role in mitochondrial ferroptosis defense. Ferroptosis is a form of regulated cell death driven by lipid peroxidation, and it has emerged as a key tumor suppression mechanism. The study shows that GPX4 (glutathione peroxidase 4) and FSP1 (ferroptosis suppressor protein 1) are two major ferroptosis defense systems. However, the study reveals that DHODH operates in parallel to mitochondrial GPX4 to inhibit ferroptosis by reducing ubiquinone (CoQ) to ubiquinol (CoQH₂), a radical-trapping antioxidant with anti-ferroptosis activity. DHODH inhibition leads to mitochondrial lipid peroxidation and ferroptosis, particularly in cancer cells with low GPX4 expression. Conversely, DHODH activation can protect cells from ferroptosis. The study further shows that DHODH inhibitors, such as brequinar, selectively suppress GPX4-low tumor growth by inducing ferroptosis. When combined with ferroptosis inducers like sulfasalazine, DHODH inhibitors synergistically induce ferroptosis and suppress GPX4-high tumor growth. These findings suggest that targeting DHODH could be a promising strategy for cancer therapy, especially in GPX4-low cancers. The study also highlights the importance of DHODH in mitochondrial lipid peroxidation and ferroptosis. DHODH is localized on the outer face of the mitochondrial inner membrane and couples the oxidation of DHO to OA with the reduction of CoQ to CoQH₂. This process is essential for suppressing ferroptosis. The study further demonstrates that DHODH inhibition can sensitize GPX4-high cancer cells to ferroptosis, while DHODH deletion induces ferroptosis in GPX4-low cancer cells. These findings suggest that DHODH is a key regulator of mitochondrial ferroptosis and that targeting DHODH could be a viable therapeutic approach for cancer treatment. The study also shows that DHODH inhibitors, such as brequinar, have potential as cancer therapeutics, especially when combined with other ferroptosis-inducing agents like sulfasalazine. Overall, the study provides new insights into the role of DHODH in ferroptosis and highlights its potential as a therapeutic target in cancer treatment.