Leukemia stem cells (LSC) represent a crucial and rare subset of cells present in acute myeloid leukemia (AML); they play a pivotal role in the initiation, maintenance, and relapse of this disease. Targeting LSC holds great promise for preventing AML relapse and improving long-term outcomes. However the precise molecular mechanisms governing LSC self-renewal are still poorly understood. Here, we present compelling evidence that the expression of miR-30e-5p, a potential tumor-suppressive microRNA, is significantly lower in AML samples than in healthy bone marrow samples. Forced expression of miR- 30e effectively inhibits leukemogenesis, impairs LSC self-renewal, and delays leukemia progression. Mechanistically, Cyb561 acts as a direct target of miR-30e-5p in LSC, and its deficiency restricts the self-renewal of LSC by activating reactive oxygen series signaling and markedly prolongs recipients' survival. Moreover, genetic or pharmacological overexpression of miR-30e-5p or knockdown of Cyb561 suppresses the growth of human AML cells. In conclusion, our findings establish the crucial role of the miR-30e-5p/Cyb561/ROS axis in finely regulating LSC self-renewal, highlighting Cyb561 as a potential therapeutic target for LSC-directed therapies.