Acute myeloid leukemia (AML) is a devastating disease characterized by poor patient outcome and suboptimal chemotherapeutics. Here, a high-throughput screen identified diosmetin, a citrus flavonoid, with anti-AML activity. Diosmetin imparted selective toxicity against leukemia and leukemia stem cells in vitro and in vivo with no effect on normal hematopoietic stem cells. Mechanistically, we demonstrated that diosmetin targets estrogen receptor (ER) β. ERβ expression conferred cell sensitivity, as patient-derived AML cells with high levels of ERβ were sensitive, whereas cells with low ERβ were insensitive to diosmetin. Knockdown of ERβ confirmed resistance, whereas overexpression enhanced sensitivity to diosmetin, which was demonstrated to be mediated by reactive oxygen species signaling. In summary, these studies highlight targeting of ERβ with diosmetin as a potential novel therapeutic strategy for the treatment of AML. Mol Cancer Ther; 16(11); 2618–26. ©2017 AACR.