The issue of how progesterone affects mammary gland growth is controversial, and the mechanism governing the effects of the hormone remains mostly unknown. We have previously shown that G protein-coupled receptor 30 (GPR30) is a progestin target gene whose expression correlates with progestin-induced growth inhibition in breast cancer cells. In this study, we investigate the role of GPR30 in regulating cell proliferation and mediating progestin-induced growth inhibition. When progestin failed to inhibit the growth of MCF-7 cells and instead stimulated growth, GPR30 was down-regulated. In this way, the inhibitory or stimulatory affects that progestin has on proliferation correlated with the level of expression of GPR30. Transient expression of GPR30 resulted in a marked inhibition of cell proliferation independent of estrogen treatment. GPR30 antisense was used to evaluate the role of GPR30 expression in progestin-induced growth inhibition. A diminished GPR30 mRNA expression by the antisense stimulated growth. Interestingly, GPR30 antisense abrogated the growth inhibitory effect of progestin and progesterone. Indeed, progestin induced 1) a reduction in cell proliferation, 2) G1-phase arrest, and 3) down-regulation of cyclin D1 was diminished. These data suggest that the orphan receptor, GPR30, is important for the inhibitory effect of progestin on growth.