C₄ plants are acclimated to drought and high temperatures that are detrimental for C₃ plants, and they are well equipped with a better antioxidant capacity to cope with oxidative stress caused by abiotic stresses. However, how C₃ and C₄ plants respond to oxidative stress that occurs in a specific compartment of the cell, such as chloroplast or mitochondria, is still not known. Therefore, understanding antioxidant responses of different carboxylation pathways to reactive oxygen species (ROS) at organelle level deserves an investigation. In the present study, the production of organelle originated ROS was induced with chemical agents specific to mitochondria (rotenone =ROT) and chloroplasts (methyl viologen = MV and DCMU), and antioxidant response of Flaveria robusta (C₃) and F. bidentis (C₄) were studied. For this purpose, activities of antioxidant system (superoxide dismutase (SOD), catalase (CAT), peroxidase (POX), ascorbate peroxidase (APX), glutathione reductases (GR)), and isoenzymes of SOD, CAT, and NADPH oxidase (NOX) activity were determined. Treatment of leaves with ROT, MV and DCMU for 4 h induced lipid peroxidation by 38%, 18% and 41% respectively in F. robusta, while 81%, 75% and 56% induction were observed for same inhibitors in F. bidentis. On the other hand, treatment with inhibitors cause decrease of CAT activity in F. robusta (27% with ROT), while increased CAT activity was observed in F. bidentis (maximum 68% with MV). Overall, the antioxidant defense responses of C₃ and C₄ plants were distinctive to organelle induced oxidative stress, especially in chloroplast-sourced accumulation of ROS.