Tropospheric ozone (O3) is a global air pollutant that causes deleterious effect to the plants. The present objective was to investigate the growth response, foliar injury, reactive oxygen species (ROS) accumulation and metabolites production in Costus pictus D. Don (insulin plant) at two developmental stages under ambient O3(AO) and ambient + 20 ppb O3(EO) using the open-top chambers (OTCs). A significant reduction in leaf area and total biomass was observed under EO as compared with AO. EO induced ROS (.O2-and H2O2) and lipid peroxidation led to more significant foliar injury and solute leakage. Image obtained from the fluorescence microscope and biochemical estimations reflected high levels of ROS under EO. A differential response in flavonoids and anthocyanin content, ascorbic acid, and antioxidative enzymes such as catalase (CAT), superoxide dismutase (SOD) and peroxidase (POX) has been observed with the growth stages of C. pictus plant. EO exposure negatively affected thiols and protein contents at all the growth stages. Secondary metabolites (tannins, lignin, saponins and alkaloids) were increased in both leaves and rhizomes due to EO, whereas phytosterols were induced only in rhizomes. Apart from other metabolites, the key bioactive compound (corosolic acid) showed its synthesis to be stimulated under EO at later growth stage. The study concludes that O3is a potent stimulating factor for changing the levels of secondary metabolites and antioxidants in an antidiabetic C. pictus plants as it can alter its medicinal properties. © 2021 CSIRO. All rights reserved.