ABSTRACTThe antioxidative enzyme monodehydroascorbate reductase (MDHAR) is represented by five genes in Arabidopsis, including four that encode cytosolic and peroxisomal proteins. The in planta importance of these specific isoforms during oxidative stress remain to be characterised. T‐DNA mutants for MDAR genes encoding cytosolic and peroxisomal isoforms were studied. To examine their roles in conditions of intracellular oxidative stress, mutants were crossed with a cat2 line lacking the major leaf catalase. Enzyme assays in mdar mutants and of recombinant MDHARs suggest that peroxisomal MDHAR1 and cytosolic MDHAR2 are major players in leaf NADH‐ and NADPH‐dependent activities, respectively. All mutants showed a wild‐type phenotype when grown in standard conditions. In the cat2 background, loss of peroxisomal MDHAR functions decreased growth whereas loss of the cytosolic MDHAR2 function had no effect on growth but annulled a large part of transcriptomic and phenotypic responses to oxidative stress. The effects of the mdar2 mutation included decreased salicylic acid accumulation and enhanced glutathione oxidation, and were reverted by complementation with the MDAR2 sequence. Together, the data show that the cytosolic MDHAR2 is dispensable in optimal conditions but essential to promote biotic defence responses triggered by oxidative stress.