From an shRNA screen, we identified ClpP as a member of the mitochondrial proteome whose knockdown reduced the viability of K562 leukemic cells. Expression of this mitochondrial protease that has structural similarity to the cytoplasmic proteosome is increased in leukemic cells from approximately half of all patients with AML. Genetic or chemical inhibition of ClpP killed cells from both human AML cell lines and primary samples in which the cells showed elevated ClpP expression but did not affect their normal counterparts. Importantly, Clpp knockout mice were viable with normal hematopoiesis. Mechanistically, we found that ClpP interacts with mitochondrial respiratory chain proteins and metabolic enzymes, and knockdown of ClpP in leukemic cells inhibited oxidative phosphorylation and mitochondrial metabolism. •ClpP is a mitochondrial protease overexpressed in a subset of AML and stem cells•Inhibition of ClpP decreases the viability of AML cells with high ClpP expression•ClpP interacts with mitochondrial respiratory chain proteins and metabolic enzymes•Genetic knockdown of ClpP impairs oxidative phosphorylation and complex II Cole et al. show that ClpP, a mitochondrial protease, is overexpressed in a large fraction of human acute myeloid leukemias (AMLs) and that inactivation of ClpP selectively kills these AML cells via inhibition of oxidative phosphorylation and mitochondrial metabolism.