Outcomes of relapsed/refractory acute myeloid leukemia (AML) are poor, and strategies to improve outcomes are urgently needed. One important factor promoting relapse and chemoresistance is the ability of AML cells to thrive in vivo within an intrinsically hypoxic bone marrow microenvironment. Here we show that human AML cells exhibit enhanced autophagy, specifically mitophagy (i.e., increased accumulation of mitochondria and decreased mitochondrial membrane potential) under hypoxia. To target this pathway, we investigated the activity of the potent chloroquine-derived autophagy inhibitor, Lys05, on human AML cells, patient samples, and patient derived xenograft models. Inhibition of autophagy by Lys05 in AML cells prevented removal of damaged mitochondria and preferentially enhanced cell death under hypoxia mirroring the marrow microenvironment. Lys05 eradicated human AML cells of all genotypes including p53 mutant cells. Lys05 treatment in primary AML xenografted mice decreased CD34+CD38- human cells and prolonged overall survival. Moreover, Lys05 overcame hypoxia-induced chemoresistance and improved the efficacy of cytarabine, venetoclax, and azacytidine in vitro and in vivo in AML models. Our results demonstrate the importance of autophagy, specifically mitophagy, as a critical survival and chemoresistance mechanism of AML cells under hypoxic marrow conditions. Therapeutic targeting of this pathway in future clinical studies for AML is warranted.