Cardiovascular disease is the leading cause of morbidity and mortality in postmenopausal women. EM-652 (acolbifene) is a fourth-generation selective ER modulator (SERM) exerting complete antiestrogenic effects on the breast and uterus. EM-652 potently inhibits bone resorption and induces positive lipid modifications in estrogen-deficient animals. As most of the cardioprotective actions of estrogen are exerted directly at the vascular level, we studied the effects of EM-652 on endothelial production of nitric oxide (NO) in vitro and in vivo. EM-652 triggers NO release by human umbilical vein endothelial cells through nongenomic mechanisms, rapidly activating endothelial nitric oxide synthase (eNOS) via an ER-dependent sequential activation of MAPKs and PI3K/Akt pathways independently from gene transcription or protein synthesis. Moreover, EM-652 increases eNOS protein levels during prolonged treatments. Upon pharmacological comparison, EM-652 is markedly more potent than the SERMs raloxifene and tamoxifen in increasing NO synthesis from endothelial cells. In ovariectomized and fertile rats, EM-652 increases aortic eNOS expression and enzymatic activity at low, but not at higher, dosages. The present data show that EM-652 (acolbifene) has estrogen-like activity on the vascular wall, directly increasing NO production through genomic and nongenomic mechanisms in vitro and in vivo.