Objective: Exclusive breastfeeding is recommended till 6 months age. Factors regulating the breastmilk iron and lactoferrin levels are incompletely known. Considering high prevalence of nutritional anemia in lactating mothers, we studied the iron status of lactating mothers, their breastmilk iron and lactoferrin levels to determine any relationship between them. Design: Prospective study with 6 months follow-up. Setting: Tertiary care referral hospital. Subjects: Hundred nonanemic and 100 anemic mothers with their babies recruited at birth. Fifty-two nonanemic and 50 anemic mothers and their babies completed the 6-month follow-up. Interventions: Hemoglobin (Hb), total iron binding capacity (TIBC), percent transferrin saturation (%TS), serum iron (SI) and serum ferritin measured on day 1 and 6 months postpartum. Breastmilk iron and lactoferrin measured on day 1, 14 weeks and 6 months after delivery. Results: Breastmilk iron decreased progressively from day 1 to 14 weeks and at 6 months in both groups, but no significant difference was noted between nonanemic and anemic mothers (P40.05). Significant decline in breastmilk lactoferrin concentration from day 1 to 14 weeks in nonanemic and anemic mothers (Po0.001) noted. Hemoglobin, TIBC, %TS, SI and serum ferritin of both groups had no correlation with breastmilk iron and lactoferrin concentration on day 1, 14 weeks and 6 months after delivery. Conclusions: Breastmilk iron and lactoferrin concentration had no relationship with the mother's Hb and iron status.