Nitrosylation is a reversible post-translational modification of protein cysteines playing a major role in cellular regulation and signaling in many organisms including plants where it has been implicated in the regulation of immunity and cell death. The extent of nitrosylation of a given cysteine residue is governed by the equilibrium between nitrosylation and denitrosylation reactions. The mechanisms of these reactions remain poorly studied in plants. In the present work, we have employed glycolytic GAPDH from Arabidopsis thaliana as a tool to investigate the molecular mechanisms of nitrosylation and denitrosylation using a combination of approaches including activity assays, the biotin switch technique, site-directed mutagenesis and mass spectrometry. Arabidopsis GAPDH activity was reversibly inhibited by nitrosylation of catalytic Cys-149 mediated either chemically with a strong NO donor or by transnitrosylation with GSNO. GSNO was found to trigger both GAPDH nitrosylation and glutathionylation although nitrosylation was widely prominent. Arabidopsis GAPDH was found to be denitrosylated by GSH but not by plant cytoplasmic thioredoxins. GSH fully converted nitrosylated GAPDH to the reduced, active enzyme, without forming any glutathionylated GAPDH. Thus, we found that nitrosylation of GAPDH is not a step towards formation of the more stable glutathionylated enzyme. GSH-dependent denitrosylation of GapC1 was found to be linked to the [GSH]/[GSNO] ratio and to be independent of the [GSH]/[GSSG] ratio. The possible importance of these biochemical properties for the regulation of Arabidopsis GAPDH functions in vivo is discussed.