Background: Estrogenic signals are suggested to have protection roles in the development of colorectal cancer (CRC). The G protein-coupled estrogen receptor (GPER) has been reported to mediate non-genomic effects of estrogen in hormone related cancers except CRC. Its expression and functions in CRC were investigated. Methods: The expression of GPER and its associations with clinicopathological features were examined. The mechanisms were further investigated using cells, mouse xenograft models, and clinical human samples. Results: GPER was significantly (p < 0.01) down regulated in CRC tissues compared with their matched adjacent normal tissues in our two cohorts and three independent investigations from Oncomine database. Patients whose tumors expressing less (n = 36) GPER showed significant (p < 0.01) poorer survival rate as compared with those with greater levels of GPER (n = 54). Promoter methylation and histone H3 deacetylation were involved in the down regulation of GPER in CRC cell lines and clinical tissues. Activation of GPER by its specific agonist G-1 inhibited proliferation, induced cell cycle arrest, mitochondrial-related apoptosis and endoplasmic reticulum (ER) stress of CRC cells. The upregulation of reactive oxygen species (ROS) induced sustained ERK1/2 activation participated in G-1 induced cell growth arrest. Further, G-1 can inhibit the phosphorylation, nuclear localization, and transcriptional activities of NF-kappa B via both canonical IKK alpha/ I kappa B alpha pathways and phosphorylation of GSK-3 beta. Xenograft model based on HCT-116 cells confirmed that G-1 can suppress the in vivo progression of CRC. Conclusions: Epigenetic down regulation of GPER acts as a tumor suppressor in colorectal cancer and its specific activation might be a potential approach for CRC treatment.