The mitochondrial enzyme dihydroorotate dehydrogenase (DHODH) catalyzes one of the rate‐limiting steps in de novo pyrimidine biosynthesis, a pathway that provides essential metabolic precursors for nucleic acids, glycoproteins, and phospholipids. DHODH inhibitors (DHODHi) are clinically used for autoimmune diseases and are emerging as a novel class of anticancer agents, especially in acute myeloid leukemia (AML) where pyrimidine starvation was recently shown to reverse the characteristic differentiation block in AML cells. Herein, we show that DHODH blockade rapidly shuts down protein translation in leukemic stem cells (LSCs) and has potent and selective activity against multiple AML subtypes. Moreover, we find that ablation of CDK5, a gene that is recurrently deleted in AML and related disorders, increases the sensitivity of AML cells to DHODHi. Our studies provide important molecular insights and identify a potential biomarker for an emerging strategy to target AML. Synopsis This study reports that AG636, an inhibitor of the metabolic enzyme DHODH, has excellent potency against acute myeloid leukemia (AML) in pre‐clinical models. AG636 exhibits potent activity against different AML subtypes in vivo, promoting a combination of cell death and differentiation and effectively reducing leukemic stem cells. DHODH inhibition has a moderate impact on normal blood development, but the effects are temporary with hematopoietic populations recovering after treatment cessation. Pyrimidine starvation limits nascent protein synthesis, in part through downregulating YY1. Loss of CDK5, a gene recurrently deleted in a subset of patients with aggressive disease, alters the molecular response of leukemic cells to AG636 and increases their sensitivity to drug treatment. This study reports that AG636, an inhibitor of the metabolic enzyme DHODH, has excellent potency against acute myeloid leukemia (AML) in pre‐clinical models.