Heme iron preparation (HIP) is a complex comprising heme iron and peptide fragments formed by enzymatic hydrolysis of hemoglobin. This complex was enriched by ultrafiltration and in heme-iron and is assumed to be water-soluble. In the present study, HIP was prepared using the blood of cultured yellowtail fish (fish-HIP) as a novel nutritional supplement. This method of preparation eliminated the need for mammalian sources. The optimal pH required for preparation of hydrophilic fish-HIP, with apparent water solubility similar to that prepared from bovine hemoglobin, was determined. An aqueous solution of fish-HIP was fractionated by centrifugation followed by filtration using membrane and ultra filters. Dynamic light scattering and ultraviolet-visible light spectroscopy demonstrated that fish-HIP was present in the hydrocolloidal form in aqueous media, with a particle size of 80-100 nm. This is the first report that demonstrated the hydrocolloidal nature of HIP. High-performance liquid chromatography (HPLC) and mass spectroscopy showed that the peptides in fish-HIP are more hydrophobic than those separated by ultra ulltration and have molecular weights ranging from 2,000 Da to 3,600 Da.