Mitochondria play an essential role in nitric oxide (NO) signal transduction in plants. Using the biotin-switch method in conjunction with nano-liquid chromatography and mass spectrometry, we identified 11 candidate proteins that were S-nitrosylated and/or glutathionylated in mitochondria of Arabidopsis (Arabidopsis thaliana) leaves. These included glycine decarboxylase complex (GDC), a key enzyme of the photorespiratory C(2) cycle in C3 plants. GDC activity was inhibited by S-nitrosoglutathione due to S-nitrosylation/S-glutathionylation of several cysteine residues. Gas-exchange measurements demonstrated that the bacterial elicitor harpin, a strong inducer of reactive oxygen species and NO, inhibits GDC activity. Furthermore, an inhibitor of GDC, aminoacetonitrile, was able to mimic mitochondrial depolarization, hydrogen peroxide production, and cell death in response to stress or harpin treatment of cultured Arabidopsis cells. These findings indicate that the mitochondrial photorespiratory system is involved in the regulation of NO signal transduction in Arabidopsis.