Background and aims: Using iron as a food additive usually causes undesirable sensory changes and side effects in humans. In this study,we made iron (Fe) nanoparticles (NPs) and studied the cytotoxicity of FeSO۴ bulk and NPs on HT-۲۹ cells and different doses of theseparticles on rat intestine.Methods: Particle size of nanoscale was achieved by mechanical technique. Iron particles were characterized using scanning electronmicroscopy (SEM) and transmission electron microscopy (TEM). The effect of iron particles with different concentrations (۶.۲۵, ۳.۱۲۵, and۱.۵۷ mM/mL) on the colon cell line was performed using the MTT assay at ۲۴, ۴۸, and ۷۲ hours. Apoptosis and necrosis of the cells wereassessed using Annexin V-FITC staining and propidium iodide (PI) at ۲۴ h. In an in vivo study, Taftoon bread was produced from fortifiedwheat flour with FeSO۴ bulk and NPs, which are recommended in human diet (۹, ۱۸, and ۲۷ mg of elemental iron/kg flour). Wistar ratswere fed daily with fortified bread for ۲۱ days and their colon and small intestine were then evaluated histopathologically. Statisticalanalyses were performed using SPSS ۲۲.۰ software by chi-square test.Results: The synthesized FeSO۴ NPs were smaller than ۱۰۰ nm, and they had more adverse effects on the viability of the HT-۲۹ cellscompared to the bulk- FeSO۴ at ۷۲ hours. Flow cytometric study showed that the early apoptosis of cells by the bulk form was more thanthe NPs, but at the low concentration (۱.۵۷ mM/mL), the NPs induced more necrosis than the bulk particles (P = ۰.۰۶۳). The survival rateof cells facing all concentrations of NPs and bulk- FeSO۴ decreased dose dependently (P = ۰.۰۷۵). In vivo results revealed that there wereno pathological changes in rats’ intestinal tissues.Conclusion: The bulk and NPs of iron have adverse effects on the HT-۲۹ cells, but no histopathological changes were seen on rats’ intestinalcells.