Eukaryotes produce a siderophore-like molecule via a remarkably conserved biosynthetic pathway. 3-OH butyrate dehydrogenase (BDH2), a member of the short-chain dehydrogenase (SDR) family of reductases, catalyzes a rate-limiting step in the biogenesis of the mammalian siderophore 2,5-dihydroxybenzoic acid (2,5-DHBA). Depletion of the mammalian siderophore by inhibiting expression of bdh2 results in abnormal accumulation of intracellular iron and mitochondrial iron deficiency in cultured mammalian cells, as well as in yeast cells and zebrafish embryos We disrupted murine bdh2 by homologous recombination to analyze the effect of bdh2 deletion on erythropoiesis and iron metabolism. bdh2 null mice developed microcytic anemia and tissue iron overload, especially in the spleen. Exogenous supplementation with 2,5-DHBA alleviates splenic iron overload in bdh2 null mice. Additionally, bdh2 null mice exhibit reduced serum iron. Although BDH2 has been proposed to oxidize ketone bodies, we found that BDH2 deficiency did not alter ketone body metabolism in vivo. In sum, our findings demonstrate a key role for BDH2 in erythropoiesis.