To determine the factors that affect milk iron content at the second week of lactation and whether the supplementation lactating mother with iron might increase breast milk iron content between 2 weeks and 4 months postpartum. Healthy mothers were enrolled 10 to 20 days postpartum, if their babies were term, normal gestational age, and exclusive breastfed. Maternal blood samples for hemoglobin (Hb), iron, iron binding capacity, serum ferritin, and breast milk samples for iron and zinc were taken and mothers were randomized into iron Supplemented Mid placebo groups. At the end of the fourth month, blood and milk samples were taken again. Forty-seven healthy mothers were included into file Study. Milk iron content was lower; however, milk-to-serum iron ratio was higher in cases with low maternal iron reserves than cases with adequate iron reserves. Mothers with low Hb level ((sic) 12g.dL) had higher milk zinc content and lower milk iron/zinc content than mothers with normal Hb value. Iron supplementation to lactating nonanemic mothers did not change milk iron content and the decline in milk iron content and milk-to-serum iron ratio. Milk iron content and milk-to-serum iron ratio of iron could be regulated by active transport in cooperation with maternal iron status.