MicroRNAs (miRNAs) are emerging as critical regulators of normal and malignant hematopoiesis. In previous studies of acute myeloid leukemia miR-9 overexpression was commonly observed. Here, we show that ectopic expression of miR-9 in vitro and in vivo significantly blocks differentiation of erythroid progenitor cells with an increase in reactive oxygen species (ROS) production. Consistent with this observation, ROS scavenging enzymes, including superoxide dismutase (Sod2), Catalase (Cat), and glutathine peroxidase (Gpx1), are down-regulated by miR-9. In addition, miR-9 suppresses expression of the erythroid transcriptional regulator FoxO3, and its down-stream targets Btg1 and Cited 2 in erythroid progenitor cells, while expression of a constitutively active form of FoxO3 (FoxO3-3A) reverses miR-9-induced suppression of erythroid differentiation, and inhibits miR-9-induced ROS production. Thus, our findings indicate that aberrant expression of miR-9 blocks erythropoiesis by deregulating FoxO3-mediated pathways, which may contribute to the ineffective erythropoiesis observed in patients with hematological malignancies.