It is known that estrogen promotes the proliferation of breast cancer cells. Agonists to P2Y 2 receptors promote or suppress proliferation in different cancers. In the present study, the methods of methylthiazoltetrazolium (MTT) assay, real-time RT-PCR, Western blot and fluorescent calcium imaging analysis were used to investigate whether P2Y 2 receptors play a role in the effects of estrogen on the breast cancer cell lines, MCF-7 and MDA-MB-231. We found that P2Y 2 receptors were expressed in both the estrogen receptor alpha (ER α )-positive breast cancer cell line MCF-7 and the ER α -negative breast cancer cell line MDA-MB-231. 17β-Estradiol (17β-E 2 ) (1pM to 1000nM) promoted proliferation of MCF-7 cells, which was blocked by the ER antagonist ICI 182,780 (1μM) and the ER α antagonist methyl-piperidino-pyrazole (MPP, 50μM), but not by the ER β antagonist 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol (PHTPP, 50μM) or ER β small interfering RNA. The P2Y 2 and P2Y 4 receptor agonist UTP (10–100μM) suppressed the viability of breast cancer cells in both MCF-7 and MDA-MB-231 cells. The effect was blocked by suramin (10–100μM), known to be an effective antagonist against P2Y 2 , but not P2Y 4 , receptor-mediated responses. 17β-E 2 played a more positive role in promoting proliferation in MCF-7 cells when suramin blocked the functional P2Y 2 receptors. 17β-E 2 (0.1–1000nM) downregulated the expression of P2Y 2 receptors in terms of both mRNA and protein levels in MCF-7 cells. The effect was blocked by ICI 182,780 and MPP, but not PHTPP or ER β small interfering RNA. 17β-E 2 did not affect the expression of P2Y 2 receptors in MDA-MB-231. UTP (10–100μM) led to a sharp increase in intracellular Ca 2+ in MCF-7 cells. Pre-incubation with 17β-E 2 (0.1μM) attenuated UTP-induced [Ca 2+ ] i , which was blocked by ICI182,780 and MPP, but not PHTPP. It is suggested that estrogen, via ER α receptors, promotes proliferation of breast cancer cells by down-regulating P2Y 2 receptor expression and attenuating P2Y 2 -induced increase of [Ca 2+ ] i .