The divalent metal ion transporter DMT1 is critical for nonheme iron import. We have previously shown that DMT1 is regulated in vitro by ubiquitination that is facilitated by the adaptor proteins Ndfip1 and Ndfip2. Here we report that in Ndfip1−/− mice fed a low- iron diet, DMT1 expression and activity in duodenal enterocytes are significant higher than in the wild-type animals. This correlates with an increase in serum iron levels and transferrin saturation. Liver and spleen iron stores were also increased in Ndfip1−/− mice fed a normal diet. Counterintuitive to the increase in iron uptake, Ndfip1−/− mice fed a low iron diet develop severe microcytic, hypochromic anemia. We demonstrate that this is due to a combination of iron deficiency and inflammatory disease in Ndfip1−/− mice, because Ndfip1−/−/Rag1−/− immunodeficient mice fed a low iron diet did not develop anemia and showed an iron overload phenotype. These data demonstrate that Ndfip1 is a critical mediator of DMT1 regulation in vivo, particularly under iron restricted conditions.