Iron overload, caused by hereditary hemochromatosis or repeated blood transfusions in some diseases, such as beta thalassemia, bone marrow failure and myelodysplastic syndrome, can significantly induce injured bone marrow (BM) function as well as parenchyma organ dysfunctions. However, the effect of iron overload and its mechanism remain elusive. In this study, we investigated the effects of iron overload on the hematopoietic stem and progenitor cells (HSPCs) from a mouse model. Our results showed that iron overload markedly decreased the ratio and clonogenic function of murine HSPCs by the elevation of reactive oxygen species (ROS). This finding is supported by the results of NAC or DFX treatment, which reduced ROS level by inhibiting NOX4 and p38MAPK and improved the long-term and multi-lineage engrafment of iron overload HSCs after transplantation. Therefore, all of these data demonstrate that iron overload injures the hematopoiesis of BM by enhancing ROS through NOX4 and p38MAPK. This will be helpful for the treatment of iron overload in patients with hematopoietic dysfunction.