Intestinal iron absorption increases during pregnancy to support maternal and fetal iron demands. Although knowledge on non-heme iron absorption has advanced substantially since identification of the hormone hepcidin in 2000, the mechanisms of heme iron absorption remain elusive. Catabolism of senescent red blood cells releases 10-times more iron into the circulation daily than does iron absorption from the diet, yet the contribution of this endogenous maternal heme iron source to fetal iron transfer is unknown. The human placenta abundantly expresses an array of heme transporters but it is unclear whether these proteins play a role in placental heme utilization. The objective of this research was to explore the mechanism and regulation of heme and non-heme iron utilization in the duodenum and the placenta, the two major sites of iron flux during pregnancy. To address these questions, stable iron isotopes (57Fe and 58Fe) were used to measure duodenal heme and non-heme iron absorption in rats and placental transfer of iron derived from maternal red blood cell catabolism and from maternal diet in pregnant women. In Sprague Dawley rats, hepcidin up-regulation suppressed the absorption of both heme and nonheme iron but the effect was more pronounced for non-heme. Hepcidin was inversely associated with iron transporters on the apical but not basolateral side of the duodenum, suggesting apical iron transport is the primary target of hepcidin action. The stable iron isotope study in pregnant women (n=16, ages 17-35 years) indicated that iron derived from maternal red blood cells was transferred to the fetus, revealing the importance of maternal red cell iron stores in supporting fetal iron demands. In a cohort of pregnant adolescents (13-18 years), placental protein expression of two putative heme transporters were associated with neonatal iron status, consistent with a role of these proteins in placental iron transport. Future research is needed to elucidate the roles of these transporters in the uptake and intracellular trafficking of heme in the placenta and to characterize the sources of heme iron in the circulation and inter-tissue heme trafficking pathways.