Technical Abstract: Adding human milk fortifiers (HMFs) to human milk (HM) is one way of overcoming the nutrient deficits found in the latter. In this study, the bioavailabilities of calcium, zinc and iron in S-26/SMA HMF added to HM were compared with those in HM fortified with various bovine milk proteins: a-lactalbumin, colostrum, caseinate, CPPs and WPC. The bioavailability of each mineral was assessed using an in vitro digestion/Caco-2 cell culture model. Calcium and zinc uptake was traced with radioisotopes; iron uptake was assessed via ferritin formation. Samples were prepared on an equal protein-content basis and with added calcium, but no zinc or iron were added. Results revealed that calcium uptake from HM+S-26/SMA was not different from any of the HM fortified with the bovine milk proteins, except for HM+colostrum and unfortified HM in which calcium uptake was significantly lower (-91 and -97%, respectively). Both zinc and iron uptakes were significantly higher for HM+S-26/SMA than for the other HM+bovine-fortifiers. This type of study enables HMF product manufacturers to balance mineral nutrition with other aspects of the HMF, such as protein content and multi-mineral bioavailability, thereby improving the nutrition, development and ultimately the survival and well-being of premature neonates.