Hemoglobin was hydrolyzed with Esperase and Flavourzyme as the endopeptidase and exopeptidase, respectively. The solubility of the heme-iron enriched peptide fraction decreased as the degree of hydrolysis of the hydrolysate increased. When the pH of a hydrolysate was adjusted to 5.0 after simultaneous hydrolysis with the two enzymes, the solubility of heme-iron enriched peptide was nearly zero, and 98% of the heme-iron enriched peptide fraction was recovered as a precipitate. These results indicated that an effective separation method for the production of heme-iron enriched peptide could be established by pH adjustment of the hemoglobin hydrolysate with high degree of hydrolysis.