We have previously shown that estrogen stimulates cell proliferation in both normal and transformed urothelial cells mainly through activation of the two primary estrogen receptors, ER and ER. A growing body of evidence suggests that estrogen also initiates nongenomic effects that cannot be explained by activation of primary estrogen receptors. In the present study, we observed that urothelial cells express high amounts of GPR30, a G protein-coupled receptor recently identified as a candidate for membrane-associated estrogen binding. Membrane- impermeable bovine serum albumin-conjugated 17-estradiol and the specific GPR30 agonist G-1 both inhibited urothelial cell proliferation in a concentration-dependent manner. Transient overexpression of GPR30 inhibited 17-estradiol-induced cell proliferation. Decreased GPR30 expression caused by specific small interfering RNA increased 17-estradiol-induced cell proliferation. These results indicate that membrane-associated inhibitory effects of 17-estradiol on cell proliferation correlates with abundance of GPR30. Although 17-estradiol induced a significant increase in caspase-3/7 activity, G-1 did not, suggesting that the GPR30-mediated inhibitory effect on cell proliferation was not caused by apoptosis. Furthermore, we found that G-1 failed to induce c-fos, c-jun, and cyclin D1 expression, and GPR30 overexpression abolished 17-estradiol-induced c-fos, c-jun, and cyclin D1 expression. However, inactivation of GPR30 by small interfering RNA increased c-fos, c-jun, and cyclin D1 expression. These results suggest that GPR30-mediated inhibition of urothelial cell proliferation is the result of decreased cyclin D1 by down-regulation of activation protein-1 signaling.