Epidemiological studies correlate moderate red wine consumption to reduced incidence of cardiovascular disease. Resveratrol is a polyphenolic compound in red wine that has cardioprotective effects in rodents. Although endothelial cell (EC) studies indicate that micromolar resveratrol has diverse biological activities, these concentrations are not physiologically relevant because human oral ingestion provides only brief exposure to nanomolar plasma levels. Previously, we reported that nanomolar resveratrol activated ERK1/2 signaling in bovine aortic ECs (BAECs). The goal of this study was to determine the mechanisms by which nanomolar resveratrol rapidly activates endothelial nitric oxide synthase (eNOS) in human umbilical vein ECs (HUVECs). We report for the first time that resveratrol increased interaction between estrogen receptor (ER), caveolin-1 (Cav-1) and c-Src, and increased phosphorylation of Cav-1, c-Src, and eNOS. Pretreatment with the lipid raft disruptor beta-methyl cyclodextrin or G inhibitor pertussis toxin blocked resveratrol- and E₂-induced eNOS activation and NO production. Depletion of endogenous ER, not ER, by siRNA attenuated resveratrol- and E₂-induced ERK1/2, Src, and eNOS phosphorylation. Our data demonstrate that nanomolar resveratrol induces ER-Cav-1-c-SRC interaction, resulting in NO production through a G-protein-coupled mechanism. This study provides important new insights into mechanisms for the beneficial effects of resveratrol in ECs.—Klinge, C. M., Wickramasinghe, N. S., Ivanova, M. M., Dougherty, S. M. Resveratrol stimulates nitric oxide production by increasing estrogen receptor -Src-caveolin-1 interaction and phosphorylation in human umbilical vein endothelial cells.