Background: Advanced age is unequivocally linked to the development of cardiovascular disease; however, the mechanisms resulting in reduced endothelial cell regeneration remain poorly understood. Here, we investigated novel mechanisms involved in endothelial cell senescence that impact endothelial cell transcription and vascular repair after injury. Methods: Native endothelial cells were isolated from young (20±3.4 years) and aged (80±2.3 years) individuals and subjected to molecular analyses to assess global transcriptional and metabolic changes. in vitro studies were conducted using primary human and murine endothelial cells. A murine aortic reendothelialization model was used to examine endothelial cell regenerative capacity in vivo. Results: RNA sequencing of native endothelial cells revealed that aging resulted in p53-mediated reprogramming to express senescence-associated genes and suppress glycolysis. Reduced glucose uptake and ATP contributed to attenuated assembly of the telomerase complex, which was required for endothelial cell proliferation. Enhanced p53 activity in aging was linked to its acetylation on K120 due to enhanced activity of the acetyltransferase MOZ (monocytic leukemic zinc finger). Mechanistically, p53 acetylation and translocation were, at least partially, attributed to the loss of the vasoprotective enzyme, CSE (cystathionine γ-lyase). CSE physically anchored p53 in the cytosol to prevent its nuclear translocation and in its absence inhibited Akt-mediated MOZ phosphorylation, which in turn increased MOZ activity and subsequently p53 acetylation. In mice, the endothelial cell–specific deletion of CSE activated p53, induced premature endothelial senescence, and arrested vascular repair after injury. In contrast, the adeno-associated virus 9–mediated reexpression of an active CSE mutant retained p53 in the cytosol, maintained endothelial glucose metabolism and proliferation, and prevented endothelial cell senescence. Adenoviral overexpression of CSE in native endothelial cells from aged individuals maintained low p53 activity and reactivated telomerase to revert endothelial cell senescence. Conclusions: Aging-associated impairment of vascular repair is partly determined by the vasoprotective enzyme CSE.