A basic leucine zipper transcription factor, NF-E2-related factor 2 (Nrf2), plays a critical role in the cellular defense mechanism by mediating a coordinate up-regulation of antioxidant responsive element-driven detoxification and antioxidant genes. Here, we report that targeted disruption of Nrf2 causes regenerative immune-mediated hemolytic anemia due to increased sequestration of damaged erythrocytes. Splenomegaly and spleen toxicity in Nrf2^-/- mice raised a possibility of hemolytic anemia and splenic extramedullary hematopoiesis in Nrf2^-/- mice. In support of this, hematology analysis revealed that Nrf2^-/- mice suffer from anemia with abnormal red cell morphologies (i.e., Howell-Jolly bodies, acantocytes, and schistocytes). In addition, Nrf2^-/- erythrocytes were more sensitive to H₂O₂-induced hemolysis, and erythrocyte-bound IgG levels were markedly increased in Nrf2^-/- mice compared with Nrf2^+/+ mice. Because IgG bound to erythrocytes in the presence of oxidative damage in erythrocytes (regardless of Nrf2 genotype), these data support that Nrf2^-/- erythrocytes have higher levels of damage compared with Nrf2^+/+ cells. Finally, Nrf2^-/- mice showed increased levels of erythrocyte-bound IgG compared with Nrf2^+/+ mice after H₂O₂ injection in vivo, suggesting that the decreased glutathione and increased H₂O₂ render the Nrf2^-/- mice more susceptible to toxicity. Taken together, these observations indicate that a chronic increase in oxidative stress due to decreased antioxidant capacity sensitizes erythrocytes and causes hemolytic anemia in Nrf2^-/- mice, suggesting a pivotal role of Nrf2-antioxidant responsive element pathway in the cellular antioxidant defense system.