In a randomized, double-blind, placebo controlled study of the effect of iron supplementation during pregnancy, iron status (hemoglobin (Hb), serum (S-)transferrin saturation, S-ferritin) and S-erythropoietin (EPO) were assessed in 120 healthy pregnant women at 14–16 weeks of gestation, and just before delivery; 63 women were treated with 66 mg iron daily, and 57 with placebo. There were no differences in baseline values in the two groups. At term, the iron treated group had significantly higher Hb, transferrin saturation, S-ferritin (median 22 μg/1 vs. 14 μg/1, (p<0.0001) and lower S-EPO compared to the placebo treated group. In the iron group, 30.2% had exhausted iron stores (i.e. S-ferritin <20 μg/1), 6.3% latent iron deficiency (S-ferritin <20 μg/1 and transferrin saturation < 15%), and no patients had iron deficiency anemia (S-ferritin <20 μg/1 and transferrin saturation < 15% and Hb < 110 g/1). In the placebo group, 93.0% had exhausted iron stores, 54.4% latent iron deficiency, and 17.5% iron deficiency anemia; S-EPO was inversely correlated to iron status markers: Hb, rs = −0.51,p<0.001; transferrin saturation, rs= −0.65,p<0.0001; S-ferritin, rs= − 0.31, R<0.01, suggesting that the elevation in S-EPO was secondary to iron deficient erythropoiesis. Newborns to iron treated mothers had higher cord S-ferritin, median 155 μg/1, than newborns to placebo treated mothers, median 118 ug/1 (p<0.02); there were no differences in birth weight, transferrin saturation, or S-EPO. Supplemental iron in a dose of 65 mg/day from the second trimester is sufficient to prevent iron deficiency in pregnant Danish women.