Acute myeloid leukemia (AML) is an aggressive and lethal blood cancer maintained by rare populations of leukemia stem cells (LSCs). Selective targeting of LSCs is a promising approach for treating AML and preventing relapse following chemotherapy, and developing such therapeutic modalities is a key priority. Here, we show that targeting telomerase activity eradicates AML LSCs. Genetic deletion of the telomerase subunit Terc in a retroviral mouse AML model induces cell-cycle arrest and apoptosis of LSCs, and depletion of telomerase-deficient LSCs is partially rescued by p53 knockdown. Murine Terc−/− LSCs express a specific gene expression signature that can be identified in human AML patient cohorts and is positively correlated with patient survival following chemotherapy. In xenografts of primary human AML, genetic or pharmacological inhibition of telomerase targets LSCs, impairs leukemia progression, and delays relapse following chemotherapy. Altogether, these results establish telomerase inhibition as an effective strategy for eliminating AML LSCs. Display omitted •Telomerase deficiency eradicates LSC function upon enforced replication•Terc−/− LSCs are eradicated via cell-cycle arrest and apoptosis•A Terc−/− LSC gene expression signature predicts an improved outcome in human AML•Imetelstat prevents the expansion of human AML LSCs in patient-derived xenografts Specific targeting of leukemia stem cells (LSCs) in acute myeloid leukemia (AML) is a promising approach for eradicating AML and preventing relapse following chemotherapy. Bruedigam et al. show that inhibiting telomerase in both mouse and human AML models targets and depletes LSCs, impairs leukemic progression, and delays relapse following chemotherapy.