Patients affected by diabetes mellitus have oxidative stress with an impaired glutathione (GSH) redox state. The objective of this study was to determine the influence of insulin on oxidative stress, defined as a reduced intracellular GSH/GSH disulfide (GSSG) ratio and lipid peroxidation by plasma thiobarbituric acid reactive substances (TBARSs) in patients with type 2 diabetes. Two experimental interventions were used: (1) measurement of GSH/GSSG ratio after insulin incubation in erythrocytes from 10 type 2 diabetic patients, and (2) measurement of intraerythrocytic GSH/GSSG ratio and plasma TBARS in 14 type 2 diabetic patients during an in vivo hyperinsulinemic condition obtained from a euglycemic hyperinsulinemic clamp study. We confirmed that our patients underwent oxidative stress as shown by the significant difference in intracellular GSH/GSSG ratio in diabetic patients as compared to controls (13.56+/-3.84 vs 27.89+/-8.37, P<.0001). We found a significant elevation in the GSH/GSSG ratio after 2 hours of incubation with insulin in erythrocytes from diabetic patients (11.56+/-1.98 to 15.61+/-2.62, P<.001). During the clamp studies, GSH/GSSG ratio had already increased after 60 minutes and even more after 120 minutes (baseline, 15.04+/-4.19; at 60 minutes, 19.74+/-6.33; at 120 minutes, 25.33+/-11.15; P<.0001). On the contrary, no significant changes were observed in plasma TBARS (3.59+/-0.77 to 3.56+/-0.83, NS). We conclude that insulin in patients with type 2 diabetes mellitus can reduce intracellular oxidative stress through increased GSH/GSSG ratio.