Protein tyrosine nitration is a post-translational modification (PTM) mediated by nitric oxide-derived molecules. Peroxisomes are oxidative organelles in which the presence of nitric oxide (NO) has been reported. We studied peroxisomal nitroproteome of pea leaves by high-performance liquid chromatography with tandem mass spectrometry (LC–MS/MS) and proteomic approaches. Proteomic analysis of peroxisomes from pea leaves detected a total of four nitro-tyrosine immunopositive proteins by using an antibody against nitrotyrosine. One of these proteins was found to be the NADH-dependent hydroxypyruvate reductase (HPR). The in vitro nitration of peroxisomal samples caused a 65% inhibition of HPR activity. Analysis of recombinant peroxisomal NADH-dependent HPR1 activity from Arabidopsis in the presence of H2O2, NO, GSH and peroxynitrite showed that the ONOO− molecule caused the highest inhibition of activity (51% at 5mM SIN-1), with 5mM H2O2 having no inhibitory effect. Mass spectrometric analysis of the nitrated recombinant HPR1 enabled us to determine that, among the eleven tyrosine present in this enzyme, only Tyr-97, Tyr-108 and Tyr-198 were exclusively nitrated to 3-nitrotyrosine by peroxynitrite. Site-directed mutagenesis confirmed Tyr198 as the primary site of nitration responsible for the inhibition on the enzymatic activity by peroxynitrite. These findings suggest that peroxisomal HPR is a target of peroxynitrite which provokes a loss of function. This is the first report demonstrating the peroxisomal NADH-dependent HPR activity involved in the photorespiration pathway is regulated by tyrosine nitration, indicating that peroxisomal NO metabolism may contribute to the regulation of physiological processes under no-stress conditions. Display omitted •Protein tyrosine nitration is a PTM mediated by NO-derived molecules.•Peroxisomal NADH-dependent HPR activity is a target of tyrosine nitration.•Tyr198 of HPR is the primary site of nitration responsible for the inhibition.