Iron plays an important role in various crucial processes in the body and its deficiency is considered currently as a serious health problem. Thus, iron supplementation strategies for both humans and animals need to be effective and safe. According to our previous studies, zinc-based nanoparticles provide safe, biodegradable, fast and efficient transport system of orally given substances to the tissues. In the current manuscript we present results of a study aimed at investigation of the ZnO nanoparticle-based Fe supplementation system (average size 100 × 250 nm). Nanostructures were orally (gavage) administered to adult mice. Animals were sacrificed at different time points with collection of blood and internal organs for analyses (tissue iron concentration, hepatic level of hepcidin, blood parameters, liver and spleen levels of ferritin, histopathology). Initial experiment was performed to compare the biological effect of doping type (Fe3+ doping vs. a mixture of Fe3+ and Fe2+). Then, the effect of acute/chronic exposure models was determined. The increase in ferritin, along with improved, crucial hematological parameters and lack of the influence on hepcidin expression indicated the chronic application of Fe3+,2+ doped ZnO nanostructures to be the most effective among tested.