Dipeptidyl peptidase-4 (DPP4) is elevated in numerous cardiovascular pathological processes and DPP4 inhibition is associated with reduced inflammation and oxidative stress. The aim of this study was to examine the role of DPP4 in endothelial senescence. Sprague-Dawley rats (24 months) were orally administrated saxagliptin (10 mg·kg·d), a DPP4 inhibitor, for 12 weeks in drinking water. Body weight, heart rate, blood glucose, and blood pressure were measured and vascular histological experiments were performed. In vitro studies were performed using HO-induced senescent human umbilical vein endothelial cells. Both in vivo and in vitro studies confirmed the elevation of DPP4 in senescent vascular endothelium, and inhibition or knockdown of DPP4 ameliorated endothelial senescence. In addition, DPP4 inhibition or silencing reduced endothelial oxidative stress levels in aging vasculature and senescent endothelial cells. Moreover, DPP4 inhibition or knockdown normalized the expression and phosphorylation of AMP-activated protein kinase-α (AMPKα) and sirtuin 1 (SIRT1) expression. Furthermore, the beneficial effects of DPP4 inhibition or knockdown on endothelial cell senescence were at least partly dependent on SIRT1 and Nrf2 activation. In conclusion, our study demonstrated that DPP4 inhibition or silencing ameliorated endothelial senescence both in vivo and in vitro by regulating AMPK/SIRT1/Nrf2. DPP4 may be a new therapeutic target to combat endothelial senescence.