Estrogen protects premenopausal women against heart disease; however, hormone replacement therapy following menopause has been linked to breast cancer and has limited benefits on the cardiovascular system. Dietary phytoestrogens, such as genistein and the metabolite equol, may serve as selective estrogen receptor (ER) modulators that prevent vascular endothelial dysfunction through enhanced generation of the vasodilator nitric oxide. The present study investigates whether equol or specific ER agonists acutely modulate intracellular signalling pathways involved in the activation of endothelial NO synthase (eNOS). Confluent cultures of human umbilical vein endothelial cells (HUVEC) were treated (100 nM, 0-10 min) with either equol, propylpyrazoletriol (PPT), an ERα agonist, or diarylpropionitrile (DPN), an ERβ agonist, in Krebs buffer containing L-arginine (100 μM) and lysates collected for Western blot analyses. Statistical signigficance of quantified data derived from n=3-6 independent cell cultures were evaluated using Student’s unpaired t tests. Immunoblotting revealed that eNOS-Ser1177 was acutely phosphorylated by equol, DPN and PPT at 2-10 min with concurrent phosphorylation of Akt and extracellular signal-regulated kinase (ERK1/2) and cAMP-responsive element binding protein (CREB). Inhibition of phosphoinositol-3-kinase/Akt by LY294002 (10 μM, 30 min) or cAMP-dependent protein kinase by H89 (1 μM, 30 min) resulted in a significant attenuation of eNOS phosphorylation by equol (2 min, 100 nM, p<0.05, n=3). Our present study provides the first direct evidence that equol can acutely phosphorylate eNOS in HUVEC via PKA, Akt and ERK1/2 activation. These acute intracellular signalling events mediate the enhanced NO generation and vascular relaxation elicited by isoflavones and specific ER agonists.