Leukemia stem cells (LSCs) are responsible for acute myeloid leukemia (AML) chemotherapy resistance and relapse. Here, we discovered that miR-34c-5p, a microRNA central to the senescence regulation network, was significantly down-regulated in AML (non-acute promyelocytic leukemia, non-APL) stem cells compared to that in normal hematopoietic stem cells (HSCs). The lower expression of miR-34c-5p in LSCs was closely correlated to the adverse prognosis and poor responses to therapy of AML patients. Increased miR-34c-5p expression induced LSCs senescence ex vivo, prevented leukemia development and promoted the eradication of LSCs in immune deficient mice. Mechanistically, forced expression of miR-34-5p induced senescence in LSCs through p53-p21-Cyclin-dependent kinase (CDK)/Cyclin or p53-independent CDK/Cyclin pathways. Exosome-mediated transfer of miR-34c-5p was one of the reasons for miR-34c-5p deficiency in LSCs. Furthermore, miR-34c-5p could increase its intracellular level by inhibiting exosome-mediated transfer via a positive feedback loop through RAB27B, a molecule that promotes exosome shedding. Overall, this study establishes a new strategy for treatment of AML patients by targeting LSCs to reinitiate senescence via increased miR-34c-5p expression. This miRNA-mediated tumor stem cell senescence could also have important therapeutic value in other malignancies.