Gastric cancer is a serious malignant tumor. Despite progression in gastric cancer research in recent years, the specific molecular mechanism underlying the pathogenesis of the disease is not completely understood. Long non-coding RNA (lncRNA) nuclear paraspeckle assembly transcript 1 (NEAT1) affects the proliferation and metastasis of multiple types of tumor cells in colorectal cancer and breast cancer but its specific role in gastric cancer requires further investigation. The aim of the present study was to analyze the role of NEAT1 in gastric cancer. The expression of endoplasmic reticulum stress marker proteins and apoptosis-related proteins in gastric cancer tissue and cell lines was analyzed using western blotting. The targeting relationship of NEAT1 and miR-500a-3p was analyzed using dual-luciferase reporter assay. Cell proliferation was analyzed using CCK8 assay and colony formation assay while cell invasion was detected using Transwell assay. Cell apoptosis was analyzed using TUNEL staining and LC3 expression through immunofluorescent staining (IF). The results showed that lncRNA NEAT1-overexpression gastric cancer cells were established to determine its effects on cell proliferation, invasion, apoptosis, autophagy and endoplasmic reticulum stress. Subsequently, microRNA (miR)-500a was overexpressed in lncRNA NEAT1-overexpression cells. Compared with the vector group, lncRNA NEAT1 overexpression significantly inhibited gastric cancer cell proliferation and invasion, but significantly promoted cell apoptosis. Furthermore, the results indicated that lncRNA NEAT1 targeted and downregulated the expression of miR-500a-3p, and miR-500a-3p targeted X-box binding protein-1 (XBP-1) mRNA. lncRNA NEAT1 overexpression-mediated inhibition of gastric cancer cell proliferation and invasion was significantly reversed by miR-500a-3p overexpression. Furthermore, compared with the vector group, the expression levels of endoplasmic reticulum stress-related proteins (XBP-1S/XBP-1U ratio and 78-kDa glucose-regulated protein) and apoptosis-related proteins (Bax and cleaved-caspase-3) were significantly upregulated by lncRNA NEAT1 overexpression; however, miR-500a-3p overexpression reversed lncRNA NEAT1 overexpression-mediated effects on protein expression. The present study demonstrated that lncRNA NEAT1 inhibited gastric cancer cell proliferation and invasion, and promoted apoptosis by regulating the miR-500a-3p/XBP-1 axis.