Glycinin, one of the predominant storage proteins in soybeans, was examined as to whether it could be used as a calcium-binding mediator after chemical phosphorylation and enzymatic hydrolysis. Glycinin is composed of six subunits. One of the proglycinin subunits $(A_{la}B_{lb})$ was overexpressed in E. coli to obtain nonphosphorylated proteins with homogeneity. To investigate the enhanced calcium-binding properties of the phosphopeptides, the proglycinin was purified, phosphorylated, and hydrolyzed with trypsin. The proglycinin expressed in E. coli was purified by ammonium sulfate precipitation, ion-exchange chromatography, and cryoprecipitation. Chemical phosphorylation by sodium trimetaphosphate was performed to obtain phosphorylated proglycinin. After the phosphorylation, one-dimensional isoelectric focusing gel electroanalysis confirmed the phosphorylation of the proglycinin. The phosphorylated peptides were then hydrolyzed with trypsin, followed by a binding reaction with calcium chloride. The calcium-bound phosphopeptides were finally separated using immobilized metal $(Ca^{2+})$ chromatography. Consequently, a limited tryptic hydrolysate of the isolated phosphopeptides exhibited an enhanced calcium-binding ability, suggesting the potential of glycinin phosphopeptides as a calcium-binding mediator with greater availability.