Estrogen appears to protect against cardiovascular disease in pre-menopausal women. However, these protective effects are absent in women with diabetes. The hyperglycemia and consequent oxidative stress observed in diabetes cause endothelial dysfunction, but specific effects on endothelial estrogen responses are not known. In this study, we hypothesized that high glucose conditions would alter the regulation of the estrogen receptors (ERs), ERalpha and ERbeta, in endothelial cells, possibly through increased oxidative stress. The role of the AMPK activator AICAR was examined on modulating the effects of high glucose. Cultured human endothelial cells were exposed to physiologically relevant doses of 17-beta-estradiol (E2) for 24h in presence of normal (5.5mM) and high (30.5mM) levels of glucose. Protein levels of estrogen receptors, ERalpha and ERbeta, were measured through western blotting. Oxidative stress was measured by the dihydroethidium (DHE) assay for superoxide. Under normal glucose, E2 increased the levels of ERalpha relative ERbeta; however, high glucose reversed the estrogen effects on endothelial ER expression. AMPK activation restored the physiological estrogen responses, likely through amelioration of oxidative stress. Control of oxidative stress by AMPK activation or anti-oxidants could restore normal estrogen responses even in presence of hyperglycemia.