Background: Supplemental vitamin D is recommended for breastfed infants in Canada at a daily dosage of 400 IU in support of bone health, and may have a role in modulating iron absorption, inflammation and hematopoiesis. Recently, a modest increase in lean body mass was observed with daily dose of 1000 IU of vitamin D in infancy. Whether this increases utilization of iron to support lean mass accretion, at a time when iron stores acquired in utero typically decline, is unknown. Objective: This secondary analysis explores whether iron status is affected by dose (1000 vs. 400 IU/day) of vitamin D supplementation during infancy. Methods: Data were from a randomized controlled trial (Montreal, Canada; March 2016 to March 2020) of 1000 (1000IU-group) vs. 400 IU (400IU-group) of vitamin D3/day in healthy term-born breastfed infants (n=49/group) with serum 25-hydroxyvitamin D <50 nmol/L. Measurements at baseline (2-6 weeks), 3, 6, and 12 months of age included: hemoglobin concentration, breastfeeding status and iron-rich food intakes. Differences in hemoglobin between groups by time were tested using linear mixed-model regression with random intercepts. Proportions were tested using Chi-square/Fisher’s Exact tests. Results: At baseline, 83.7% of infants were exclusively breastfed with no differences in proportions between groups thereafter. At 3 months, infant formula had been introduced to 25% of the 1000IU-group and 20.5% of the 400IU-group. By 6 months, infant formula and/or iron-rich foods had been introduced (1000IU-group: 97.6% vs. 400IU-group: 92.5%). Baseline hemoglobin concentration (mean±SE) was 138±2 g/L. Hemoglobin was lower at 3 months in the 1000IU-group (106±1 vs. 111±2 g/L, P<0.005), but not thereafter (6 months 1000IU-group: 112±1 vs. 400IU-group: 113±1 g/L, P=0.34; 12 months 1000IU-group: 113±2 vs. 400IU-group: 110±2 g/L, P=0.10) and sex differences were not observed. At 3 months, the proportion with anemia (<110 g/L) was not different between groups (P=0.07); however the proportion with moderate anemia (<100 g/L) was higher in the 1000 IU-group (25% vs. 4.7%, P=0.014) and hemoglobin concentration inversely related to lean mass (beta=-0.4 per kg lean mass, P=0.028). Conclusions: Iron stores acquired in utero may be transiently depleted in infants by 3 months of age in association with lean mass accretion and a daily dosage of 1000 IU of vitamin D. Lack of differences between vitamin D dosages thereafter is likely ascribed to intakes of iron-rich infant formula and/or foods. Future biomarkers include C-reactive protein, alpha-1-acid glycoprotein, ferritin and soluble transferrin receptors to better understand vitamin D-iron-interactions in infants.