Gastric cancer (GC) is the third leading cause of cancer-related mortality worldwide. Oxysterol-binding protein-related protein 8 (ORP8) functions as a sterol sensor that regulates a number of cellular functions. We showed that ORP8 expression was significantly lower in GC tissues and cells. Overexpression of ORP8 significantly inhibited GC cell proliferation in several GC cells. The formation of colonies in AGS cells was inhibited by the overexpression of ORP8. Moreover, overexpression of ORP8 significantly decreased implanted tumor growth in nude mice. Overexpression of ORP8 resulted in a significant increase in CHOP and GRP78 expression and the phosphorylation of PERK, indicating the occurrence of ER stress. Inhibition of ER stress by 4-PBA notably suppressed overexpression of ORP8-induced decrease of GC cell proliferation, formation of colonies, and implanted tumor growth. Overexpression of ORP8 resulted in a significant decrease in Wnt3a and β-catenin expression, and activation of Wnt signaling by HLY78 markedly blocked overexpression of ORP8-induced decrease in GC cell proliferation, formation of colonies, and implanted tumor growth. 4-PBA inhibited overexpression of ORP8-induced decrease in Wnt signaling. Furthermore, overexpression of ORP8 resulted in significant activation of mitochondrial apoptotic events and increase in apoptosis, which was inhibited by 4-PBA and HLY78. Induction of ER stress, inhibition of Wnt signaling, and apoptotic cell death were involved in ORP8-induced inhibition of GC cell proliferation. These findings indicate that downregulation of ORP8 plays a pivotal role in the progression of GC, and it may be a novel therapeutic target in the treatment of GC.