A　novel　peptide　with　a　specific　calcium-binding　capacity　was　isolated　from　whey　protein　hydrolysates.　The　isolation　procedures　included　diethylaminoethyl　(DEAE)　anion-exchange　chromatography,　Sephadex　G-25　gel　filtration,　and　reversed-phase　high-performance　liquid　chromatography　(HPLC).　A　peptide　with　a　molecular　mass　of　237.99　Da　was　identified　by　liquid　chromatography-electrospray　ionization/mass　spectrometry　(LC-ESI/MS),　and　its　amino　acid　sequence　was　confirmed　to　be　Gly-Tyr.　The　calcium-binding　capacity　of　Gly-Tyr　reached　75.38　μg/mg,　increasing　by　122%　when　compared　to　the　hydrolysate　complex.　The　chelating　interaction　mode　between　the　Gly-Tyr　and　calcium　ion　was　investigated,　indicating　that　the　major　binding　sites　included　the　oxygen　atom　of　the　carbonyl　group　and　nitrogen　of　the　amino　or　imino　group.　The　folding　and　structural　modification　of　the　peptide　arose　along　with　the　addition　of　the　calcium　ion.　The　profile　of　1H　nuclear　magnetic　resonance　(NMR)　spectroscopy　demonstrated　that　the　electron　cloud　density　around　the　hydrogen　nucleus　in　the　peptide　changed　was　caused　by　the　calcium　ion.　The　results　of　ζ　potential　showed　that　the　Gly-Tyr-Ca　chelate　was　a　neutral　molecule　in　which　the　calcium　ion　was　surrounded　by　the　specific　binding　sites　of　the　peptide.　Moreover,　thermogravimetry-differential　scanning　calorimetry　(TG-DSC)　and　calcium-releasing　assay　revealed　that　the　Gly-Tyr-Ca　chelate　exerted　excellent　thermal　stability　and　solubility　in　both　acidic　and　basic　conditions,　which　were　beneficial　to　calcium　absorption　in　the　gastrointestinal　tract　of　the　human　body　and,　therefore,　improved　its　bioavailability.　These　findings　further　the　progress　in　the　research　of　whey　protein,　suggesting　the　potential　in　making　peptide-calcium　chelate　as　a　dietary　supplement.　©　2014　American　Chemical　Society.