The G protein-coupled receptor GPR30 (GPER) binds 17β-estradiol (E2), yet differs from classical estrogen receptors (ERα and ERβ). GPR30 can mediate E2-induced non-genomic signaling, but its role in ERα-positive breast cancer remains unclear. Gene expression microarray data from 5 cohorts comprising 1,250 breast carcinomas showed an association between increased GPR30 expression and ERα-positive status. We therefore examined GPR30 in estrogenic activities in ER-positive MCF-7 breast cancer cells using G-1 and diethylstilbestrol, ligands which selectively activate GPR30 and ER, respectively, and small interfering RNAs (siRNAs). In expression studies, E2 and diethylstilbestrol but not G-1 down-regulated both ER and GPR30, indicating this was ER mediated. In Ca2+ mobilization studies, GPR30 but not ERα mediated E2-induced Ca2+ responses, since E2, 4-hydroxytamoxifen (also activates GPR30) and G-1, but not DES, elicited cytosolic Ca2+ increases not only in MCF-7 cells, but also in ER-negative SKBr3 cells. Additionally, in MCF-7 cells, GPR30 depletion blocked E2- and G-1-induced Ca2+ mobilization, but ERα depletion did not. Interestingly, GPR30-coupled Ca2+ responses were sustained and inositol triphosphate receptor-mediated in ER-positive MCF-7 cells, but transitory and ryanodine receptor-mediated in ER-negative SKBr3 cells. Proliferation studies involving GPR30 depletion indicated that GPR30's role was to promote SKBr3 cell growth, but reduce MCF-7 cell growth. Supporting this, G-1 profoundly inhibited MCF-7 cell growth, potentially via p53 and p21 induction. Further, flow cytometry showed that G-1 blocked MCF-7 cell cycle progression at the G(1)-phase. Thus, GPR30 antagonizes growth of ERα-positive breast cancer, and may represent a new target to combat this disease.