The transcription factor, p53, and the adaptor protein, p66shc, both play essential roles in promoting oxidative stress in the vascular system. However, the relationship between the two in the context of endothelium-dependent vascular tone is unknown. Here, we report a novel, evolutionarily conserved, p53-mediated transcriptional mechanism that regulates p66shc expression and identify p53 as an important determinant of endothelium-dependent vasomotor function. We provide evidence of a p53 response element in the promoter of p66shc and show that angiotensin II-induced upregulation of p66shc in endothelial cells is dependent on p53. In addition, we demonstrate that downregulation of p66shc expression, as well as inhibition of p53 function in mice, mitigates angiotensin II-induced impairment of endothelium-dependent vasorelaxation, decrease in bioavailable nitric oxide, and hypertension. These findings reveal a novel p53-dependent transcriptional mechanism for the regulation of p66shc expression that is operative in the vascular endothelium and suggest that this mechanism is important in impairing endothelium-dependent vascular relaxation.