Epoetin beta pegol (C.E.R.A.) is a novel, long-acting, erythropoiesis-stimulating agent. We investigated iron utilization associated with erythropoiesis, and how it is affected by the long-term suppression of hepcidin following C.E.R.A. treatment in mice. C57BL/6N mice were administered a single intravenous injection of C.E.R.A. Hemoglobin (Hb) levels were significantly elevated following C.E.R.A. administration, while serum hepcidin levels were continuously suppressed. Expression levels of duodenal iron transporters, ferroportin (FPN) which is internalized by hepcidin binding and divalent metal transporter 1 (DMT1), increased after C.E.R.A. administration. To evaluate the contribution of dietary iron absorption to erythropoiesis following C.E.R.A. treatment, C.E.R.A.-treated mice were fed either an iron-deficient diet to restrict dietary iron absorption or a control diet. The restriction of dietary iron absorption resulted in impaired Hb elevation after C.E.R.A. administration. To investigate the utilization of stored iron for erythropoiesis following C.E.R.A. treatment, iron indices were analyzed in C.E.R.A.-treated mice given an intraperitoneal pre-treatment injection of iron-dextran. Serum hepcidin levels and splenic hemosiderin deposition decreased after C.E.R.A. administration. These results indicate that C.E.R.A. promotes utilization of iron for erythropoiesis both through enhancement of dietary iron absorption and mobilization of iron storage from reticuloendothelial cells.