Gastric cancer (GC) is a major global health problem. Chemotherapy is a common therapeutic strategy for cancers including GC. However, chemoresistance strikingly limits the clinical applications of chemotherapeutic drugs. Long non-coding RNAs (lncRNAs) have been widely reported to be implicated in the pathogenesis and chemoresistance of cancers including GC. Our work aims to investigate the roles and molecular mechanisms of lncRNA gastric cancer-associated transcript 1 (GACAT1) in regulating doxorubicin (ADR) and vincristine (VCR) resistance in GC. In this text, RT-qPCR assay showed that GACAT1 expression was markedly reduced in ADR- or VCR-resistant GC (SGC7901/ADR or SGC7901/VCR) cells and GC tissues. CCK-8 assay and flow cytometry analysis revealed that GACAT1 overexpression alleviated the resistance of GC cells to ADR and VCR. RT-qPCR and western blot assay disclosed that GACAT1 deactivated the AKT/mTOR/S6K1 signaling pathway and promoted PTEN expression in SGC7901/ADR or SGC7901/VCR cells. Restoration experiments demonstrated that GACAT1 attenuated ADR or VCR resistance by regulating the PTEN/AKT/mTOR/S6K1 pathway in SGC7901/ADR or SGC7901/VCR cells. In vivo experiments demonstrated that GACAT1 overexpression inhibited tumor growth and enhanced ADR- or VCR-mediated anti-tumor effects in GC xenograft tumor models. Taken together, these data revealed that GACAT1 weakened the resistance of GC cells to ADR and VCR by the PTEN/AKT/mTOR/S6K1 regulatory pathway in vitro and in vivo, shedding new light on GACAT1 upregulation as a potential strategy to alleviate chemoresistance in GC.