Objective Hyperglycemia contributes to the development and progression of diabetic complications; however, molecular mechanisms underlying the development of vasculopathy and potential strategies for prevention have not been identified. Methods We investigated the mechanism of hyperglycemia-induced vascular endothelial apoptosis, and the potential benefits of C-peptide replacement therapy through AMP-activated protein kinase α (AMPKα)-dependent mechanism, using human umbilical vein endothelial cells and aorta of diabetic mice. Results High glucose (33 mM) increased intracellular ROS through a mechanism involving inter-regulation between cytosolic and mitochondrial ROS generation in endothelial cells. C-peptide prevented high glucose -induced ROS generation, mitochondrial fission, mitochondrial membrane potential collapse, and endothelial cell apoptosis, through the activation of AMPKα. Additionally, the AMPK activator AICAR and the anti-hyperglycemic drug metformin mimicked protective effects of C-peptide. C-peptide replacement therapy through subcutaneously implanted osmotic pump normalized hyperglycemia-induced AMPKα dephosphorylation, ROS generation, and mitochondrial disorganization in aorta of diabetic mice. Conclusion These findings highlight a novel mechanism by which C-peptide activates AMPKα and protects against hyperglycemia-induced ROS generation, mitochondrial fission, and endothelial cell apoptosis, and may provide a new approach for preventing diabetic vascular complications.