The identification of S-nitrosated substrates and their target cysteine residues is a crucial step to understand the signaling functions of nitric oxide (NO) inside the cells. Here, we show that the key nitrogen metabolic enzyme glutamine synthetase (GS) is a S-nitrosation target in Medicago truncatula and characterize the molecular determinants and the effects of this NO-induced modification on different GS isoenzymes. We found that all the four M. truncatula GS isoforms are S-nitrosated, but despite the high percentage of amino acid identity between the four proteins, S-nitrosation only affects the activity of the plastid-located enzymes, leading to inactivation. A biotin-switch/mass spectrometry approach revealed that cytosolic and plastid-located GSs share an S-nitrosation site at a conserved cysteine residue, but the plastidic enzymes contain additional S-nitrosation sites at non-conserved cysteines, which are accountable for enzyme inactivation. By site-directed mutagenesis, we identified Cys369 as the regulatory S-nitrosation site relevant for the catalytic function of the plastid-located GS and an analysis of the structural environment of the SNO-targeted cysteines in cytosolic and plastid-located isoenzymes explains their differential regulation by S-nitrosation and elucidates the mechanistic by which S-nitrosation of Cys369 leads to enzyme inactivation. We also provide evidence that both the cytosolic and plastid-located GSs are endogenously S-nitrosated in leaves and root nodules of M. truncatula, supporting a physiological meaning for S-nitrosation. Taken together, these results provide new insights into the molecular details of the differential regulation of individual GS isoenzymes by NO-derived molecules and open new paths to explore the biological significance of the NO-mediated regulation of this essential metabolic enzyme.