Abstract Osteopontin (OPN) is a multifunctional bioactive protein that is implicated in numerous biological processes such as bone remodeling, inhibition of ectopic calcification, and cellular adhesion and migration, as well as several immune functions. Osteopontin has cytokine-like properties and is a key factor in the initiation of T helper 1 immune responses. Osteopontin is present in most tissues and body fluids, with the highest concentrations being found in milk. In the present study, ELISA for human and bovine milk OPN were developed and OPN concentration in human breast milk, bovine milk, and infant formulas was measured and compared. The OPN concentration in human milk was measured to approximately 138mg/L, which corresponds to 2.1% (wt/wt) of the total protein in human breast milk. This is considerably higher than the corresponding OPN concentrations in bovine milk (∼18mg/L) and infant formulas (∼9mg/L). Moreover, bovine milk OPN is shown to induce the expression of the T helper 1 cytokine IL-12 in cultured human lamina propria mononuclear cells isolated from intestinal biopsies. Finally, the OPN concentration in plasma samples from umbilical cords, 3-mo-old infants, and pregnant and nonpregnant adults was measured. The OPN level in plasma from 3-mo-old infants and umbilical cords was found to be 7 to 10 times higher than in adults. Thus, the high levels of OPN in milk and infant plasma suggest that OPN is important to infants and that ingested milk OPN is likely to induce cytokine production in neonate intestinal immune cells.