The impact of 100 μg ml alumina (AlO) nanoparticles (NPs) on (fenugreek) in vitro cultures was studied within 3 weeks (on days 1, 7, 14, and 21) and compared with the control and bulk (macrometer-sized particles) alumina-treated groups. Transmission electron microscopy (TEM) and dynamic light scattering were used for the characterization of NPs. The results of TEM analysis represented a nearly spherical shape for the NPs with agglomeration. The zeta potential of alumina NPs was - 25.4 ± 2.5 mV and the averaged diameter was 20 ± 5 nm. Atomic absorption and inductively coupled plasma-optical emission spectroscopy provided evidence for the release and uptake of aluminum. Treatment of cultures with NPs led to an increase in the formation of lateral roots. Treatment of fenugreek with bulk alumina caused a significant decrease in the number of leaves on day 21 (< 0.05) and the root length on days 14 and 21 compared with the control group (< 0.05). Alumina NP has led to a significant increase in the malondialdehyde content on days 7, 14, and 21 (< 0.001). The glutathione content was decreased significantly in NP and bulk-treated groups on days 1 and 7 (< 0.05). FRAP assay results showed that NPs and bulk alumina led to a decrease in the antioxidant power on days 7, 14, and 21 (< 0.001). The increased activity of catalase (< 0.001) and ascorbate peroxidase (< 0.001) was observed in the bulk-treated group. Lignin content had a significant increase in response to NPs on days 14 and 21 (< 0.001). Conclusively, alumina nano/macro particles affected agronomical and physiological properties of ; however, smaller sized particles do not necessarily imply greater toxicity, while uptake of the aluminum ions should be considered seriously.