We have recently shown that aorta from streptozotocin-(STZ)-induced diabetic rats and A10 vascular smooth muscle cells (VSMC) exposed to high glucose exhibited decreased levels of Gi proteins. In the present studies we investigated the implication of oxidative stress in hyperglycemia/ diabetes-induced decreased expression of Gi protein and adenylyl cyclase signaling in VSMC by using antioxidants. The levels of Gi proteins were significantly decreased in A10 VSMC exposed to high glucose and in aortic VSMC from STZ-diabetic rats as compared to control cells and were restored to control levels by antioxidants. In addition, MnTBAP and uric acid, scanvergers of peroxynitrite and N⁶-nitro-L-arginine methylester (L-NAME); an inhibitor of nitric oxide synthase (NOS) but not catalase also restored high glucose-induced decreased expression of Gi proteins to the control levels in A10 VSMC. Furthermore, the enhanced production of superoxide anion (O₂^-) and increased activity of NADPH oxidase in these cells were also restored to control levels by DPI, an inhibitor of NADPH oxidase. In addition, the diminished inhibition of adenylyl cyclase activity by inhibitory hormones and forskolin (FSK)-stimulated adenylyl cyclase activity by low concentrations of GTPS as well as enhanced stimulation of adenylyl cyclase by stimulatory agonists in hyperglycemic cells were restored to control levels by antioxidant treatments. These results suggest that high glucose-induced decreased levels of Gi proteins and associated signaling in A10 VSMC may be attributed to the enhanced oxidative stress due to augmented levels of peroxynitrite and not to H₂O₂.