Background: Osteopontin (OPN) is an important breastmilk protein involved in infant intestinal, immunological, and brain development. However, little is known about how common milk pasteurization and storage techniques affect this important bioactive protein. Methods: Human milk osteopontin concentration was measured in single-donor fresh or frozen breastmilk, pooled Holder-pasteurized donor breastmilk, and a shelf-stable (retort pasteurized) breastmilk product by ELISA. Breastmilk samples were pasteurized and/or frozen before measuring osteopontin concentrations. Results: Holder pasteurization of breastmilk resulted in a around 50% decrease in osteopontin levels within single-donor samples, whereas pooled donor breastmilk had comparable osteopontin levels to non-pasteurized single-donor samples. Breastmilk from mothers of preterm infants trended toward higher osteopontin concentration than mothers of term infants; however, samples from preterm mothers experienced greater osteopontin degradation upon pasteurization. Finally, freezing breastmilk prior to Holder pasteurization resulted in less osteopontin degradation than Holder pasteurization prior to freezing. Conclusion: Commonly used breastmilk pasteurization and storage techniques, including freezing, Holder and retort pasteurization, decrease the levels of the bioactive protein osteopontin in human breastmilk.