The hepcidin-ferroportin axis crucially determines systemic iron homeostasis. Hepcidin (Hpc), a 25-aminoacid peptide secreted by the liver, negatively regulates the only known iron exporter ferroportin (Fpn) to reduce iron absorption and mobilization, this way balancing iron demands (1). Liver Hpc mRNA expression is modulated in response to hypoxia, anaemia, iron levels, inflammation and erythropoiesis (2), but the interactions between these signals are not well understood. This study aims to elucidate the primary signal(s) regulating Hpc expression during chronically elevated erythropoiesis. To this end, we investigated iron homeostasis in male wild type (Wt, C57BL/6, n=4) animals and in transgenic mice (Tg6, (TgN(PDGFBEPO)321ZbZ), n=5) that chronically overexpress human erythropoietin (12-fold compared to Wt) resulting in haematocrit values of about 80%. In addition, we treated Wt (Wt_DXT, n=4) and Tg6 (Tg6_DXT, n=3) animals with a single dose of iron dextran (5 mg administered intraperitoneally), and reduced erythropoiesis in Tg6 (Tg6_SPL, n=2) by splenectomy (3). Surgery was performed under isoflurane anaesthesia (5% induction, 2% maintenance, 100% oxygen) and animals were treated with 0.075 mg/kg of buprenorphin to provide analgesia. All the procedures were approved by the Kantonales Veterinäramt. Real time-rtPCR showed that liver Hpc mRNA expression was significantly suppressed in Tg6 (p<0.001 vs. Wt). Iron administration resulted in a 5-fold increase in Hpc expression in Wt_DXT mice (p=0.024 vs. Wt) and enhanced Hpc expression in Tg6_DXT animals to levels comparable to untreated Wt mice. In addition, Hpc expression was elevated in Tg6_SPL compared to Tg6 (p<0.01), but remained low compared to Wt (p=0.002). Western blotting for the divalent metal transporter-1 revealed no changes in protein levels at the mucosal side of the duodenum. On the other hand, at the basolateral side, Fpn was upregulated in Tg6 mice (3-fold vs. Wt, p=0.047) and reduced to normal levels in Tg6_DXT and Tg6_SPL. Iron mobilization from liver and spleen was not altered, though Wt_DXT showed a decrease of Fpn level in the spleen (p=0.048 vs. Wt). The potential to acquire iron, as measured by western blotting of the transferrin receptor-1 (TfR1), showed a dramatic increase in Tg6 vs. Wt mice in liver (p<0.001) and spleen (p=0.003). In Tg6_DXT animals TfR1 was reduced to normal levels in liver (p<0.001 vs. Tg6), but remained elevated in the spleen (p=0.007 vs. Wt; ns vs. Tg6). On the other hand, splenectomy decreased TfR1 expression in the liver (p=0.006) whereas it remained elevated compared to Wt (p=0.026). Finally, L and H ferritin (Ft) subunits were increased in Wt_DXT and Tg6_DXT, whilst in Tg6_SPL only H-Ft was elevated (p=0.04 vs. Tg6; ns vs. Wt). Additionally, L-Ft but not H-Ft increased in Tg6_DXT in the spleen (p<0.001 vs. Tg6; ns vs. Wt). Overall, the data we obtained so far suggest that the body’s iron content overrides the erythropoietic signal regarding Hpc regulation and, thus, is the primary signal affecting iron homeostasis during chronically elevated erythropoiesis.