P2Y receptors are expressed in airway epithelia and play a significant role in regulating transepithelial ion transport. P2Y receptors belong to the family of purinergic receptors, which can be stimulated by nucleotides such as UTP and UDP. P2Y receptors are G protein-coupled receptors and classically signal through Gq, resulting in an increase in intracellular Ca2+ concentration ([Ca2+]i). Moreover, P2Y receptors have been implicated in asthmatic inflammation. Recent data suggest that estrogen (17β-estradiol, E2) is an important hormone that protects the lungs from inflammatory damage. In addition to the classical nuclear hormone receptors ERα and ERβ, a novel estrogen receptor, G protein-coupled estrogen receptor (GPER/GPR30), was recently identified and found to be involved in both rapid signalling and transcriptional regulation. The action of GPR30 is unclear, but it has been implicated in mediating anti-inflammatory responses. Our study aimed to investigate the inhibitory effect of GPR30 or E2 receptor activation on P2Y receptor-mediated Ca2+ signalling pathway and cytokine production in human bronchial epithelia. In this study, a human bronchial epithelial cell line, 16HBE14o-, derived from human bronchial surface epithelial cells was used. In some experiments, primary normal human bronchial epithelial cells were also used (ScienCell Research Laboratories, San Diego, CA, USA). mRNA and protein expression of GPR30 and classical estrogen receptors were examined by real-time PCR and Western blotting. Localization of GPR30 was examined by immuofluorescence staining and Western blotting of fractionated cell lysates. Data in our study demonstrate that both primary normal human bronchial epithelial cells and the 16HBE14o- cell line express GPR30 at the mRNA and protein levels, as demonstrated by real-time PCR and western blotting, respectively. Expression of GPR30 receptors was localized in the human bronchial epithelial cells by immunofluorescence staining and Western blotting of fractionated cell lysates. [Ca2+]i was examined using a calcium imaging technique in Fura-2-loaded cells. Stimulation of epithelial cells with E2 or with the specific agonist of GPR30, G1, rapidly attenuated a UDP- or UTP-evoked increase in [Ca2+]i, while this effect was reversed by GPR30 specific antagonist, G15. E2 or G1 also inhibited the secretion of two pro-inflammatory cytokines, interleukin (IL)-6 or IL-8, in cells stimulated by different nucleotides, including UDP, UTP and ATPγS. Taken together, our data suggest that the anti-inflammatory role of GPR30 may be due to its opposing effect on the pro-inflammatory pathway activated by the P2Y receptors in inflamed airway epithelia.