Estrogen classically drives lung cancer development via estrogen receptor beta (ER beta). However, fulvestrant, an anti-estrogen-based endocrine therapeutic treatment, shows limited effects for non-small cell lung cancer (NSCLC) in phase II clinical trials. G protein-coupled estrogen receptor (GPER), a third estrogen receptor that binds to estrogen, has been found to be activated by fulvestrant, stimulating the progression of breast, endometrial, and ovarian cancers. We here demonstrated that cytoplasm-GPER (cGPER) (80.49 %) and nucleus-GPER (53.05 %) were detected by immunohistochemical analysis in NSCLC samples. cGPER expression was related to stages IIIA-IV, lymph node metastasis, and poorly differentiated NSCLC. Selective agonist G1 and 17 beta-estradiol (E-2) promoted the GPER-mediated proliferation, invasion, and migration of NSCLC cells. Additionally, in vitro administration of E-2 and G1 increased the number of tumor nodules, tumor grade, and tumor index in a urethane-induced adenocarcinoma model. Importantly, the pro-tumorigenic effects of GPER induced by E-2 were significantly reduced by co-administering the GPER inhibitor G15 and the ER beta inhibitor fulvestrant, as compared to administering fulvestrant alone both in vitro and in vivo. Moreover, the phosphorylation of MAPK and Akt was involved in E-2/G1-induced GPER activation. In conclusion, our results indicated that a pro-tumor function of GPER exists that mediated E-2-/G1-dependent NSCLC progression and showed better efficiency regarding the co-targeting of GPER and ER beta, providing a rationale for further investigation of anti-estrogen clinical therapy.