Triple-negative breast cancers lack estrogen receptor α (ERα), progesterone receptor, and do not overexpress human epidermal growth factor receptor 2 (Her-2). They are neither susceptible to endocrine therapy nor to a therapy using the anti-Her-2 antibody, trastuzumab. Therefore, an efficient targeted therapy is warranted. Triple-negative breast tumors frequently express membrane bound estrogen receptor G-protein coupled receptor (GPR30). As proof of principle, we analyzed the consequences of a knock-down of GPR30 expression on the growth regulation of triple-negative breast cancer cell lines. Cells of triple-negative breast cancer cell lines were transfected with siRNA against GPR30 or control siRNA, and cell growth was stimulated either with 10(⁻⁹) M 17β-estradiol or 10(⁻⁶) M 4-hydroxytamoxifen. Cell proliferation was measured using Alamar blue staining. Activation of c-Src and epidermal growth factor (EGF)-receptor was assessed using western blot. Expression of c-fos was quantified by reverse transcription polymerase chain reaction. Seven days after transfection with siRNA, GPR30 mRNA in triple-negative breast cancer cell lines MDA-MB-435 and HCC1806 was reduced by 74 and 90%, respectively. 10(⁻⁸) M 17β-estradiol enhanced proliferation of MDA-MB-435 to 129.6±5.4% of control (p<0.05) and HCC1806 to 156.9±15.4% of control (p<0.05), respectively. 10(⁻⁶) M 4-hydroxytamoxifen increased cell number of MDA-MB-435 to 121.0±6.9% of control (p<0.05) and HCC1806 to 124.5±12.1% of control (n.s.), respectively. This increased proliferation by the two estrogenic compounds was completely prevented by knock-down of GPR30 expression in both cell lines. In control cells, activity of Src kinase was increased 3-fold by estradiol and 3.8-fold using 4-hydroxytamoxifen. Transactivation of the EGF-receptor was similarly increased in both cell lines by 17β-estradiol and 4-hydroxytamoxifen. Both compounds increased c-fos expression 1.5- and 3.1-fold, respectively. Knock-down of GPR30 expression completely abolished activation of all these signaling pathways responsible for enhanced proliferation. A pharmacological inhibition of GPR30 by specific small molecular inhibitors might prove to be an appropriate targeted therapy of triple-negative breast cancer in the future.