Heme-regulated eIF2alpha kinase (HRI) is essential for regulating globin translation in iron deficiency and in beta-thalassemia. We investigated the role of heme-regulated eIF2alpha kinase in hemoglobin and red blood cell production as well as in iron homeostasis in a mouse model of iron overload. We show that HRI deficiency does not significantly affect red cell parameters of hemochromatosis (HFE(-)(/)(-)) mice. Importantly, heme-regulated eIF2alpha kinase deficiency exacerbates decreases in hepcidin expression and splenic macrophage iron in HFE(-)(/)(-) mice. Furthermore, the serum level of bone morphogenic protein 2, which positively regulates hepcidin, is reduced in heme-regulated eIF2alpha kinase deficiency, but not in HFE deficiency.