Epigallocatechin gallate (EGCG), a green tea polyphenol, promotes vasodilation by phosphatidylinositol 3-kinase-dependent activation of Akt and endothelial nitric oxide synthase to stimulate production of nitric oxide. Reduction in endothelin-1 (ET-1) synthesis may also increase bioavailability of nitric oxide. We hypothesized that the phosphatidylinositol 3-kinase-dependent transcription factor FOXO1 may mediate effects of EGCG to regulate expression of ET-1 in endothelial cells. EGCG treatment (10 microm, 8 h) of human aortic endothelial cells reduced expression of ET-1 mRNA, protein, and ET-1 secretion. We identified a putative FOXO binding domain in the human ET-1 promoter 51 bp upstream from the transcription start site. Trans-activation of a human ET-1 (hET-1) promoter luciferase reporter was enhanced by coexpression of a constitutively nuclear FOXO1 mutant, whereas expression of a mutant FOXO1 with disrupted DNA binding domain did not trans-activate the hET-1 promoter. Disrupting the hET-1 putative FOXO binding domain by site-directed mutagenesis ablated promoter activity in response to overexpression of wild-type FOXO1. EGCG stimulated time-dependent phosphorylation of Akt (S(473)), FOXO1 (at Akt phosphorylation site T(24)), and AMP-activated protein kinase alpha (AMPK alpha) (T(172)). EGCG-induced nuclear exclusion of FOXO1, FOXO1 binding to the hET-1 promoter, and reduction of ET-1 expression was partially inhibited by the AMPK inhibitor Compound C. Basal ET-1 protein expression was enhanced by short interfering RNA knock-down of Akt and reduced by short interfering RNA knock-down of FOXO1 or adenovirus-mediated expression of dominant-negative Foxo1. We conclude that EGCG decreases ET-1 expression and secretion from endothelial cells, in part, via Akt- and AMPK-stimulated FOXO1 regulation of the ET-1 promoter. These findings may be relevant to beneficial cardiovascular actions of green tea.