Acute myeloid leukemia (AML) cells have high oxidative phosphorylation and mitochondrial mass and low respiratory chain spare reserve capacity. We reasoned that targeting the mitochondrial RNA polymerase (POLRMT), which indirectly controls oxidative phosphorylation, represents a therapeutic strategy for AML. POLRMT-knockdown OCI-AML2 cells exhibited decreased mitochondrial gene expression, decreased levels of assembled complex I, decreased levels of mitochondrially-encoded Cox-II and decreased oxidative phosphorylation. POLRMT-knockdown cells exhibited an increase in complex II of the electron transport chain, a complex comprised entirely of subunits encoded by nuclear genes, and POLRMT-knockdown cells were resistant to a complex II inhibitor theonyltrifluoroacetone. POLRMT-knockdown cells showed a prominent increase in cell death. Treatment of OCI-AML2 cells with 10-50 µM 2-C-methyladenosine (2-CM), a chain terminator of mitochondrial transcription, reduced mitochondrial gene expression and oxidative phosphorylation, and increased cell death in a concentration-dependent manner. Treatment of normal human hematopoietic cells with 2-CM at concentrations of up to 100 µMdid not alter clonogenic growth, suggesting a therapeutic window. In an OCI-AML2 xenograft model, treatment with 2-CM (70 mg/kg, i.p., daily) decreased the volume and mass of tumours to half that of vehicle controls. 2-CM did not cause toxicity to major organs. Overall, our results in a preclinical model contribute to the functional validation of the utility of targeting the mitochondrial RNA polymerase as a therapeutic strategy for AML.