A calcium-binding peptide was isolated from the hydrolysates of Korean cow serum protein (KCSP). KCSP was hydrolyzed using three different types of proteases, Alcalase, Flavourzyme, and Protamex, and the degree of hydrolysis was determined and monitored using trinitrobenzenesulfonic acid and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The hydrolysates of KCSP using Alcalase were selected and ultra-filtered below 3 kDa. The membrane-filtered solution was then fractionated using ion exchange chromatography and normal phase high-performance liquid chromatography to isolate a calcium-binding peptide. The calcium-binding capacity was determined by the orthophenanthroline method. The sequence of the purified calcium-binding peptide was analyzed using liquid chromatography/electron spray ionization (LC/ESI)-tandem mass spectroscopy and identified to be Asp-Asn-Leu-Pro-Asn-Pro-Glu-Asp-Arg-Lys-Asn-Tyr-Glu, which has a molecular weight of 1,603 Da. In addition, isolation of iron and calcium-binding peptides derived from cottonseed meal protein(CMP) hydrolysates was investigated. The degree of hydrolysis of CMP by Flavourzyme was monitored using the trinitrobenzensulfonic acid method and sodium dodecyl sulfate-polyacrylamide ge electrophoresis. The enzymatic hydrolysis of CMP for 12 hr was sufficient for the preparation of CMP hydrolysates, and the hydrolysates were filterd under 3 kDa as a molecular weight. The filterd solution was fractionated using Q-Sepharose fast flow, Sephadex G-15, and reversed phase-high performance liquid chromatography for iron and calcium-binding peptides. As a result, F51 fraction was obtained as the best candidate for calcium and iron chelation. In conclusion, the isolated iron and calcium-binding peptides can be used as functional food additives, similar to a iron and calcium supplements.