The mechanisms underlying the inactivation of Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase (G6PDH) induced by peroxyl radicals (ROO^●) and peroxynitrite (ONOO⁻), were explored. G6PDH was incubated with AAPH (2,2′ -azobis(2-methylpropionamidine)dihydrochloride), used as ROO^● source, and ONOO⁻. Enzymatic activity was assessed by NADPH generation, while oxidative modifications were analyzed by gel electrophoresis and liquid chromatography (LC) with fluorescence and mass detection. Changes in protein conformation were studied by circular dichroism (CD) and binding of the fluorescent dye ANS (1-anilinonaphthalene-8-sulfonic acid). Incubation of G6PDH (54.4 μM) with 60 mM AAPH showed an initial phase without significant changes in enzymatic activity, followed by a secondary time-dependent continuous decrease in activity to ∼59% of the initial level after 90 min. ONOO⁻ induced a significant and concentration-dependent loss of G6PDH activity with ∼46% of the initial activity lost on treatment with 1.5 mM ONOO⁻. CD and ANS fluorescence indicated changes in G6PDH secondary structure with exposure of hydrophobic sites on exposure to ROO^●, but not ONOO⁻. LC-MS analysis provided evidence for ONOO⁻-mediated oxidation of Tyr, Met and Trp residues, with damage to critical Met and Tyr residues underlying enzyme inactivation, but without effects on the native (dimeric) state of the protein. In contrast, studies using chloramine T, a specific oxidant of Met, provided evidence that oxidation of specific Met and Trp residues and concomitant protein unfolding, loss of dimer structure and protein aggregation are involved in G6PDH inactivation by ROO^●. These two oxidant systems therefore have markedly different effects on G6PDH structure and activity.