Bienertia sinuspersici performs single cell C-4 photosynthesis without Kranz anatomy. Peripheral and central cytoplasmic compartments in a single chlorenchyma cell act as mesophyll cells and bundle sheath cells. Development of this specialized mechanism is gradual during plant development. Young leaves perform C-3 photosynthesis, while mature leaves have complete C-4 cycle. The aim of this work was to investigate changes in redox regulation and antioxidant defence during transition from C-3 to single cell C-4 photosynthesis in B. sinuspersici leaves. First, we confirmed gradual development of C-4 with protein blot and qRT-PCR analysis of C-4 enzymes. After this activities and isoenzymes of superoxide dismutase (SOD), catalase (CAT), peroxidase (POX), ascorbate peroxidase (APX), glutathione reductase (GR), dehydroascorbate reductase (DHAR) and H2O2 and TBARS and glutathione pool and redox status (GSH/GSSG) were determined in young, developing and mature leaves during transition from C-3 to single cell C-4 photosynthesis. Activities of SOD, APX and POX decrease, while GR and DHAR were increased. However, most striking results were the changes in isoenzyme patterns of SOD, CAT and GR which were gradual through transition to C-4 photosynthesis.