Zinc oxide nanoparticles (NPs) are the third highest in terms of global production among the various inorganic nanoparticles, and there are concerns because of their worldwide availability and accumulation in the environment. In contrast, zinc is an essential element in plant growth and metabolism, and ZnO NPs (nano-ZnO) may have unknown interactions with plants due to their small sizes as well as their particular chemical and physical characteristics. The present study examined the effect of nano-ZnO (25nm) and bulk or natural form (<1000nm, bulk-ZnO), compared with zinc in the ionic form (ZnSO4) on Nicotiana tabacum seedlings in a nutrient solution supplemented with either nano-ZnO, bulk-ZnO (0.2, 1, 5 and 25µM) or ZnSO4 (control) for 21 days. Results showed that nano-ZnO at most of the levels and 1µM bulk-ZnO positively affected growth (root and shoot length/dry weight), leaf surface area and its metabolites (auxin, phenolic compounds, flavonoids), leaf enzymatic activities (CAT, APX, SOD, POX, GPX, PPO and PAL) and anatomical properties (root, stem, cortex and central cylinder diameters), while bulk-ZnO caused decreases at other levels. The activities of enzymes were induced to a greater extent by intermediate nano-ZnO levels than by extreme concentrations, and were higher in nano-ZnO treated than in bulk treated tobacco. As the ZnO level increased, the vascular expansion and cell wall thickening of the collenchyma/parenchyma cells occurred, which was more pronounced when treated by NPs than by its counterpart. The Zn content of root and leaf increased in most of ZnO treatments, whereas the Fe content of leaves decreased. Our findings indicate that tobacco responded positively to 1µM bulk-ZnO and to nearly all nano-ZnO levels (with the best levels being at 0.2µM and 1µM) by morphological, physiological and anatomical adjustments.