Iron metabolism during pregnancy is biased towards maintaining the fetal supply, even at the cost of anaemia in the mother. The mechanisms regulating this are not well understood. Here we examine iron deficiency and supplementation on the hierarchy of iron supply and the gene expression of proteins which regulate iron metabolism in the rat. Dams were fed iron deficient diets for four weeks, mated and either continued on the deficient diet or an iron supplemented diet during either half of pregnancy. A control group were maintained on normal iron throughout. They were killed at 0.5, 12.5 or 21.5 days of gestation and tissues and blood samples collected. Samples were also collected from fetuses at day 21.5 of gestation. Deficiency and supplementation had differential effects on maternal and fetal haematocrit and liver iron levels. From early in pregnancy a hierarchy of iron supply is established benefiting the fetus to the detriment of the mother. Transferrin receptor, transferrin receptor 2 and hepcidin mRNA expression were regulated by iron deficiency and supplementation. Expression patterns showed both organ and supplementation protocol dependence. Further analysis indicated that iron levels in the fetal, and not maternal, liver regulate the expression of liver transferrin receptor and hepcidin expression in the mother.