Hypertension is a major contributor to cardiovascular mortality, which remains the leading cause of death. Reduced arteriolar lumen due to increased proliferation of smooth muscle cells (SMCs) could precipitate hypertension. Although SMCs from large vessels have been extensively studied, very little is known about mechanisms regulating proliferation of arteriolar SMCs, particularly in response to 17β estradiol (estrogen). We hypothesized that estrogen inhibits proliferation of arteriolar SMCs by activating exchange protein activated by cAMP (EPAC). Cells were isolated from dermal arterioles of healthy subjects using 2% lidocaine as local anesthetic, after written informed consent. Statistical analysis was done using student’s t-test for comparing two means. Data are reported as means ± SEM. We show that estrogen significantly inhibits proliferation of arteriolar SMCs in a concentration- (10-11-10-7 M) and time- (0-96 hours) dependent fashion. A 37 ± 3.9 % inhibition in proliferation was observed after 48 hours of treatment with 10-9 M of estrogen (n=3; p<0.01). Pretreatment with the estrogen receptor (ER) antagonist ICI-182,780 (ICI; 10-8 M) significantly (n=3; p<0.05) reduced estrogen’s inhibition (39±4 % inhibition with estrogen versus 21±3% inhibition with ICI + estrogen). Moreover, transfection with ER alpha or beta potentiated estrogen’s effect (61±7 or 47±4% inhibition with ER alpha or beta respectively; n=3 and p<0.01 for either). Treatment of cells with the cell-impermeable form of estrogen, estrogen:BSA (10-8 M) also caused a significant 27.3 ± 4.1% inhibition in proliferation, suggesting that a membrane estrogen receptor mediates the estrogen effect (p<0.05). Moreover, pretreatment with the adenylate cyclase inhibitor SQ-22536 (400µM) abolished the estrogen effect, indicating that cAMP mediates the noted estrogen effect. Additionally, we show that the cAMP analogue, 8-(4-chloro-phenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate (0-150 µM) which selectively activates EPAC, mimics the antiproliferative effects of estrogen. PKA does not appear to be involved since pretreatment with the PKA inhibitor (H89; 5µM) did not significantly affect the estrogen-induced effect (100% (no H89) versus 111 ± 4.7% (+H89); p>0.05). Transfection with a dominant negative mutant of EPAC (EPAC-R279E) reduced estrogen’s effect (39 ± 2.1 versus 22 ± 3.2 %; p<0.05), suggesting that EPAC mediates the observed estrogen effect. Taken together, our data show that estrogen elicits its antiproliferative effect via a Gs-coupled membrane ER, which elevates cAMP levels leading to EPAC activation. This novel effect of estrogen could partially account for its protective role in premenopausal women compared to age-matched men, particularly in regard to hypertension incidence.