This study evaluated the influence of iron supplementation in pregnancy and breastfeeding on iron status of lactating women from a Brazilian Human Milk Bank. Blood and mature breast milk samples were collected from 145 women for assessment of iron status, as well as copper and zinc status. Haemoglobin, serum iron and ferritin were determined, respectively, by electronic counting, colorimetry and chemiluminescence. Transferrin and ceruloplasmin were analysed by nephelometry. Serum copper and zinc were measured by atomic absorption spectrophotometry, and serum alkaline phosphatase was measured by a colorimetric method. Iron, zinc and copper in breast milk were determined by spectrometry. Mean values of iron, copper and zinc (blood and breast milk) were compared by ANOVA, followed by Tukey's test. Iron supplementation was beneficial to prevent anaemia in pregnancy but not effective to treat anaemia. During breastfeeding, iron supplementation had a negative effect on maternal copper status, confirming an interaction between these micronutrients.