In this study, the impact of Fe 3 O 4 nanoparticles (NPs) at different concentrations was studied on physiology, antioxidant enzymes, cell damage, and secondary metabolites of Physalis alkekengi seedlings under in vitro conditions. Seeds were grown on a solid MS medium containing different Fe 3 O 4 NPs concentrations (0, 10, 20, and 30 mg/L). The results showed that Fe 3 O 4 NP at 20 mg/L increased growth (fresh and dry weight, length of shoot and root, and the number of adventitious roots) and physiological (relative water content, chlorophyll pigments, carotenoid, carbohydrate, H 2 O 2 , and ion leakage) parameters through stimulation of protein content and antioxidant enzyme activities of catalase (CAT), superoxide dismutase (SOD), and ascorbate peroxidase. Fe 3 O 4 NP at 20 mg/L was found to be the optimum concentration for enhancing secondary metabolites (total flavonoids, phenolics, and withanolides) and activity of DPPH radical scavenging and PAL, while the higher Fe 3 O 4 NPs concentration (30 mg/L) showed toxicity impact on growth parameters through promoting H 2 O 2 level, lipoxygenase activity, and ion leakage and inactivation of the antioxidant enzyme isoforms. The electrophoretic pattern displayed Fe 3 O 4 NPs at 30 mg/L decreased the band intensity of SOD (Mn-SOD and Cu/Zn-SOD1) and CAT (CAT1 and 2) isoforms. These findings display that the optimal dose of Fe 3 O 4 NPs (20 mg/L) acts as an elicitor to induce antioxidant functions of P. alkekengi cell system to suppress oxidative damage.