Objective:　The　main　purpose　of　this　research　is　separation　and　structural　property　characterization　of　calcium-binding　peptide　from　whey　protein　hydrolysate.　Methods:　The　purification　procedure　contained　anion-exchange　chro-matography　on　DEAE-650M,　gel　filtration　chromatography　on　Sephadex　G-25　and　RP-HPLC.　Calcium-binding　activity　was　measured　after　every　separation　procedure,　and　NMR,　XRD,　TG-DSC,　Zeta　potential　were　used　to　analyze　its　calcium-binding　properties.　Results:　A　peptide　having　high　calcium-binding　capacity　was　isolated　from　whey　protein　hydrolysate,　named　WPH-13,　whose　activity　was　63.49　μg/mg.　Structure　identification　showed　that　the　calcium　perhaps　was　wrapped　by　one　or　several　WPH-13,　main　incorporation　sites　were　carboxylate　oxygen　and　amino　nitrogen.　The　pH　stability,　thermostability　and　antioxidant　activity　have　also　improved.　Conclusion:　A　calcium-binding　peptide　can　be　isolated　from　whey　protein　hydrolysate　and　the　structural　property　identification　was　used　to　study　the　chelating　mechanism,　which　laid　a　foundation　of　production　application　of　the　third　calcium-supplemental　agents.　©　2015,　Chinese　Institute　of　Food　Science　and　Technology.　All　right　reserved.