This study explores the antioxidative effect of a specific wheat germ-derived peptide on high glucose-induced oxidative stress in vascular smooth muscle cells (VSMCs) and the underlying mechanisms. The peptide ADWGGPLPH was identified by LC-MS/MS. The effects of this peptide on the production of ROS and the expression of oxidative stress signaling proteins in VSMCs were determined. STZ-induced mice were utilized to confirm the anti-oxidative and anti-diabetic cardiovascular disease effects of this peptide in vivo. The results showed that ADWGGPLPH significantly prevented high glucose-induced cell proliferation, decreased intracellular ROS generation, stimulated AMPK activity, inhibited the PKCζ, AKT and Erk1/2 phosphorylation, and suppressed NOX4 protein expression. In addition, ADWGGPLPH enhanced the antioxidant abilities and attenuated inflammatory cytokine generation in STZ-induced diabetic mice. Therefore, ADWGGPLPH prevents high glucose-induced oxidative stress in VSMCs by modulating the PKCζ/AMPK/NOX4 pathway.