Background: There were some non-linier doses, low usefulness and disadvantages resulted from epidemiological studies using Estrogen Replacement therapy as prevention or active treatment in coronary Vascular Diseases. In vitro studies also showed that the increase of the dose of Estrogen used it was not increase the diameter of the vessels. Therefore the aim of this study was to elucidate the effect of 17 β Estradiol induce NO (nitric oxide) in endothelial dysfunction of HUVEC (human umbilical vein endothelial cell) culture, and also its effect in the denuded and intact aorta of guinea pig. Method : This study used experimental design to observe the effect of 17 β Estradiol in vasodilatation aorta ring of female guinea pigs and the concentration of NO released from normal and dysfunction endothelial of HUVECs culture. Endothelial dysfunction was done by exposed high glucose (33mM) to HUVECs culture. The study used non genomic (8 minutes) and genomic (30, 60, 120 and 240 minutes) effect of 17 β Estradiol (10-8M). To observe the vasodilatation and the concentration of NO, bioassay technique was used. Results: The results showed that there was an increase of NO release from HUVEC culture exposed to high glucose when incubated by 17 β Estradiol (10-8M), but this increase of NO never reached the normal value. In the intact aorta was approved that 17 β Estradiol (10-8M) was more higher the ability of relaxation compare to the denuded aorta. Conclusion: it is concluded that non genomic effect of 17 β Estradiol (10-8M) shows the domination of endothelial role to increase the activation of NO regard to the vasodilatation effect.