The ability of casein phosphopeptides (CPPs) to bind and transport minerals has been previously studied. However, the single bioactive peptides responsible for the effects of CPPs have not been identified. This study was to purify calcium-binding peptides from CPPs and to determine their effects on calcium and magnesium uptake by Caco-2 cell monolayers. Five monomer peptides designated P1 to P5 were isolated and the amino acid sequences were determined using LC-MS/MS. Compared with the CPP-free control, all five monomeric peptides exhibited significant enhancing effects on the uptake of calcium and magnesium (P < 0.05). Interestingly, when calcium and magnesium were presented simultaneously with P5, magnesium was taken up with priority over calcium in the Caco-2 cell monolayers. For example, at 180 min, the amount of transferred magnesium and calcium was 78.4 ± 0.95 μg/well and 2.56 ± 0.64 μg/well, respectively, showing a more than 30-fold difference in the amount of transport caused by P5. These results provide novel insight into the mineral transport activity of phosphopeptides obtained from casein.