Acute myeloid leukemia (AML) is one of the most common hematologic malignancies, thus large amounts of patients relapse and succumb to the disease. In this study, we investigated the effects of stigmasterol on leukemia both in vitro and in vivo. We identified a critical dependence of AML blasts on cholesterol for proliferation and morphogenesis. Stigmasterol treatment induced apoptosis and caused G1/S cell cycle arrest in leukemia cells. Furthermore, stigmasterol effectively suppressed AML progression in vivo. Mechanistically, stigmasterol supplementation resulted in reduced intracellular cholesterol levels, along with decreased expression of sterol regulatory element-binding protein 2 (SREBP2) and its downstream effectors. These findings indicate that stigmasterol inhibits leukemia cell proliferation and promotes apoptosis by disrupting SREBP2-dependent cholesterogenesis in leukemia cells. Collectively, our study reveals a crucial role for stigmasterol in suppressing AML progression through apoptosis induction and highlights the potential mechanisms of SREBP2-mediated abnormal cholesterogenesis in regulating AML.