The soy phytoestrogen, genistein, has an array of biological actions, including weak estrogenic effects, inhibition of tyrosine kinase, and cellular antioxidant activity. Recent studies showed that genistein may improve vascular function, but the mechanism is unclear. We show that genistein stimulates intracellular cAMP accumulation in intact bovine aortic endothelial cells and human umbilical vein endothelial cells over an incubation period of 30 min. Increases in intracellular cAMP are evoked by as low as 10 nM genistein but not by estrogen. These increases in cAMP may result primarily from enhanced adenylate cyclase activity by a mechanism that does not involve genomic actions or estrogen receptors. The cAMP induced by genistein activates cAMP-dependent protein kinase (PKA) in bovine aortic endothelial cells. The activation of PKA phosphorylates and activates cAMP response element binding protein, leading to up-regulation of cAMP response element-containing gene expression. In addition, activation of PKA protects thrombin-induced endothelial monolayer permeability, a novel cardioprotective effect of genistein mediated by the cAMP/PKA cascade. These findings demonstrate that a nongenomic action of genistein leads to activation of the cAMP/PKA signaling system to protect the vascular barrier function and alter the expression of cAMP-regulated genes, thereby providing a novel mechanism underlying some of the cardiovascular protective effects proposed for soy phytoestrogens.