G protein-coupled estrogen receptor (GPER) is recently identified as a membrane-associated estrogen receptor that mediates non-genomic effects of estrogen. Our previous immunohistochemistry study found an association between GPER and the proliferation of epithelial ovarian cancer. However, the contributions and mechanisms of GPER in the proliferation of ovarian cancers are not clear. We have examined the role of GPER in estrogen receptor α (ERα)-negative/GPER positive OVCAR5 ovarian cancer cell line. MTT assay was used to detect cell proliferation. BrdU incorporation assay was used to measure the cells in S-phase. Protein expression of marker genes of proliferation, cell cycle and apoptosis were examined by Western blot. The results showed that 17β-estradiol and selective GPER agonist G-1 stimulated the proliferation of OVCAR5 cells and increased the cells in S-phase. Both ligands upregulated the protein levels of c-fos and cyclin D1. Small interfering RNA targeting GPER or G protein inhibitor pertussin toxin (PTX) inhibited basal cell proliferation and attenuated 17β-estradiol- or G-1-induced cell proliferation. GPER mediated cell growth was also associated with the apoptosis of OVCAR5 cells. These findings suggest that GPER has an important function in the proliferation of ovarian cancer cells lacking ERα. GPER might be a promising therapeutic target in ovarian cancer.