The use of ⁵⁷Fe-isotopically enriched ferritin for the accurate measurement of Fe : ferritin ratios is proposed for metabolic studies. Thus, the synthesis of ⁵⁷Fe-isotopically enriched ferritin from horse apo-ferritin and isotopically enriched (NH₄)₂⁵⁷Fe(II)(SO₄)₂ (Mohr's salt) is conducted. Size exclusion chromatography on-line with UV-VIS absorption (at 380 nm) is used in order to monitor the loading process of apo-ferritin. These studies revealed that the Fe-incorporation process involves also the formation of protein aggregates (oligomers) showing higher molecular mass than ferritin. A final optimized protocol involving incubation of the synthesized standard with guanidine hydrochloride (pH 3.5) has provided the best conditions for maintaining a stable protein structure without aggregates. Such ⁵⁷Fe-isotopically enriched ferritin was characterized and contained an average of 2200 atoms of Fe per mole of ferritin. The evaluation of the Fe-core after saturation with ⁵⁷Fe by Transmission Electron Microscopy (TEM) has revealed the formation of ⁵⁷Fe nanoparticles with a similar diameter to that of the commercial Fe-containing ferritin, confirming the process of Fe uptake, oxidation and mineralization within the protein cavity. The synthesized ⁵⁷Fe-ferritin shows great potential as a nanometabolic tracer to study the kinetics of Fe release in the cases of iron metabolic disorders.