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In order to utilize fish byproducts in a calcium supplement with high solubility, hoki (Johnius belengerii) frames composed of flesh and skeleton discarded from industrial processing were degraded by pepsin in acetic acid solution (pH 2.2). After digestion, a calcium-binding peptide was isolated from the pepsinolytic hydrolysates using a hydroxyapatite affinity chromatography. Calcium-binding assay elucidated that J. belengerii frame peptide (JFP) can solubilize a similar amount of calcium with casein phosphopeptide (CPP). In ESI-QTOF tandem mass analysis for peptide identification, the amino acid sequence of JFP showed high similarity to those of actin (NCBInr database), was identified as Val-Leu-Ser-Gly-Gly-Thr-Thr-Met-Tyr-Ala-Ser-Leu-Tyr-Ala-Glu (MW: 1,561 Da).