Type 2 diabetes mellitus (T2DM) is a major risk factor for cardiovascular diseases. The reduction of mitochondrial protein sirtuin protein 3 (SIRT3) has been reported to contribute to the development of T2DM by impacting mitochondrial respiration. Cordycepin is an adenosine derivative and is isolated from the culture filtrate of Cordyceps militaris. This study explored the protective effect of cordycepin on vascular impairment induced by T2DM and its properties and protective mechanism. In this study, a T2DM rat model was established. The endothelium-dependent relaxation of the thoracic aorta ring decreased in T2DM rats could be reversed by cordycepin. Next, mitochondrial impairment in human umbilical vein endothelial cells was detected by JC-1 staining. In vitro studies revealed that cordycepin plays a beneficial role in advanced glycation end product-induced endothelial mitochondrial impairment. Moreover, according to the cordycepin molecular docking analysis, cordycepin can bind to SIRT3. Cordycepin increased the expression and activation of SIRT3 in a dose-dependent manner. SIRT3 interruption blocked the protective effect of cordycepin on mitochondria in human umbilical vein endothelial cells. Cordycepin can conclusively protect vascular function impaired by T2DM, and the mechanism may potentially be involved in SIRT3 signaling pathways.