Phytoestrogens have been implicated as promising therapeutic agents to treat the vascular impairment seen in menopausal women. The present study investigated the long-term effects of phytoestrogens from Curcuma comosa Roxb. on vascular relaxation of isolated thoracic aorta from ovariectomized (OVX) rats. Treatment of OVX rats for 12 weeks with C. comosa powder, hexane extract, and a novel phytoestrogen, diarylheptanoid-D3, [(3R)-1,7-diphenyl-(4E,6E)-4,6- heptadien-3-ol] prevented impairment of the endothelium-dependent relaxation response to acetylcholine in OVX, but not the endothelium-denude aortic ring relaxation in response to sodium nitroprusside. These data suggest that the vascular relaxation effect of C. comosa is mediated via endothelial cells. Treatment with D3 also increased endothelial nitric oxide synthase (eNOS) and estrogen receptor-α (ERα) protein expression in the aorta of OVX rats and suppressed elevated tumor necrosis factor-α (TNF-α) expression in OVX aortic rings. These results indicate that C. comosa treatment prevents impairment of vascular relaxation in estrogen-deficient animals via the ER-eNOS pathway as well as through its ability to promote an anti-inflammatory response. © 2011 American Chemical Society.