BACKGROUND: Human milk is rich in osteopontin (OPN), which has immunomodulatory functions. METHODS: In a randomized controlled trial, standard formula (SF) and the same formula with 65 mg of OPN/L (F65) or 130 mg of OPN/L (F130), representing similar to 50 and 100% of the OPN concentration in human milk, were compared. We examined frequencies and composition of peripheral blood immune cells by four-color immunoflow cytometry of formula-fed infants at ages 1, 4, and 6 months, and compared them with a breastfed (BF) reference group. RESULTS: The F130 group had increased T-cell proportions compared with the SF (P = 0.036, average effect size 0.51) and F65 groups (P = 0.008, average effect size 0.65). Compared with the BF group, the monocyte proportions were increased in the F65 (P=0.001, average effect size 0.59) and F130 (P=0.006, average effect size 0.50) groups, but were comparable among the formula groups. CONCLUSION: OPN in an infant formula at a concentration close to that of human milk increased the proportion of circulating T cells compared with both SF and formula with added OPN at similar to 50% of the concentration in human milk. This suggests that OPN may favorably influence immune ontogeny in infancy and that the effects appear to be dose-dependent.