The iron nanoparticles with different physic-chemical properties induce inconsistent effects on various studied plant species. Thus, the effect of ferric oxide (Fe2O3) nanoparticles was compared with Fe2O3 microparticles and FeSO4complexes of EDTA for major physiological and gene expression in Rue (Ruta graveolens). Iron root content increased as Fe-MPs + EDTA ˂˂ Fe-NPs + EDTA˂ FeSO4 + EDTA. The shoot's iron remained unchanged or slightly increased under most of FeSO4 and Fe-MPs + EDTA treatments. Under Fe-NPs + EDTA treatment, 50 and 250 µM concentration decreased on shoot iron by 23.2% and 19.4% compared to control, respectively. But the shoot iron at 500 µM NPs was 28.2% higher than that of the control. A 46-58 fold lower Fe translocation was observed under Fe-NPs + EDTA than Fe-MPs + EDTA. The effect of Fe-NPs + EDTA was more significant on plant fresh and dry mass than the control. All treatments showed an increase in anthocyanin by 19-84% in leaves compared to the control. The Fe-NPs + EDTA and MPs + EDTA induced similar effects on enhanced growth parameters, total chlorophyll, catalase enzyme activity, gene, and reduced chlorophyll a/b and oxidants. Catalase enzyme activity in FeSO4 and MPs + EDTA was similar, and in Fe-NPs + EDTA treatments were influenced by coarse and fine regulation mechanisms, respectively. Iron MPs + EDTA had a more negative effect on IRT1 relative gene expression in roots as compared to other iron forms. The IRT1 relative gene expression in shoots was positively affected by 31-81% under all treatment types (except control and 250 µM Fe-NPs + EDTA, and 250 µM MPs + EDTA). These results could reveal the potential mechanism of plant response to nanoparticles.