Peroxiredoxins (Prxs) are a family of antioxidant proteins that reduce peroxide levels by using reducing agents such as thioredoxin. These proteins were characterized to have a number of cellular functions, including cell proliferation and differentiation and protection of specific proteins from oxidative damage. However, the physiological roles of the peroxiredoxins have not been determined. To clarify the physiological relevance of this protein type, we generated a mouse model deficient in Prx II, which is abundantly expressed in all types of cells. The Prx II^–/– mice were healthy in appearance and fertile. However, they had splenomegaly caused by the congestion of red pulp with hemosiderin accumulation. Heinz bodies were detected in their peripheral blood, and morphologically abnormal cells were elevated in the dense red blood cell (RBC) fractions, which contained markedly higher levels of reactive oxygen species (ROS). The Prx II^–/– mice had significantly decreased hematocrit levels, but increased reticulocyte counts and erythropoietin levels, indicative of a compensatory action to maintain hematologic homeostasis in the mice. In addition, a labeling experiment with the thiol-modifying reagent biotinylated iodoacetamide (BIAM) in Prx II^–/– mice revealed that a variety of RBC proteins were highly oxidized. Our results suggest that Prx II^–/– mice have hemolytic anemia and that Prx II plays a major role in protecting RBCs from oxidative stress in mice.