Both pregnancy and carrying multiple fetuses increase the iron (Fe) demands across gestation. Adequate Fe stores during pregnancy are essential to support maternal physiological changes and fetal growth. However, currently there are few data available on Fe homeostasis in this high-risk obstetric and neonatal population. This research aimed to provide normative data on maternal and neonatal anemia and Fe status in the context of multiple gestations and identify significant determinants of maternal or neonatal anemia. Maternal Fe status, inflammatory status, and folate and vitamin B-12 status were monitored across pregnancy (~24 wks, n=73) and at delivery (~35 wks, n=61) in 83 women (aged 20-46 y) carrying multiple fetuses. Neonatal umbilical cord blood samples and placental tissue were obtained from 183 neonates at delivery (25-38 weeks of gestation) to assess neonatal Fe status, inflammation status, and folate and vitamin B-12 status. A high prevalence of maternal anemia and iron deficiency (ID) was evident in this group of women carrying multiple fetuses. The prevalence of maternal tissue ID (as measured by serum transferrin receptor) increased significantly from pregnancy to delivery. Maternal erythropoietin (EPO) at either mid-gestation or at delivery was found to be the most sensitive predictor of maternal anemia at delivery. These results suggest that additional screening and Fe supplementation may be warranted in women carrying multiple fetuses. In this study population, 14% of neonates were anemic at birth. Neonates born to women who were obese prior to pregnancy or smoked cigarettes during pregnancy had significantly greater odds of having anemia at birth, and screening for Fe status in these neonates may be warranted. At this time there are no specific guidelines for Fe status screening in this population and neonates are highlighted for additional screening only if they are born prematurely. Hepcidin, a systemic regulator of Fe homeostasis, was detectable in umbilical cord blood from nearly all neonates born between 25-38 weeks of gestation in this study. At present, it is not clear when the fetus begins to produce hepcidin, these data add to the normative information available on this topic. In addition, cord hepcidin may regulate cord Fe status independently from maternal hepcidin, as significant associations were only found between cord hepcidin and cord Fe status. Cord hepcidin had a greater intrauterine variance than interuterine variance and significantly impacted the intrauterine variances in cord Hb, serum ferritin, soluble transferrin receptor and EPO, suggesting that cord hepcidin plays an important role in regulation of fetal Fe homeostasis.