The use of microsatellite instability (MSI) and mismatch repair (MMR) as predictive biomarkers for fluorouracil-based adjuvant chemotherapy in colorectal cancer has been a paradigm shift. However, whether this applies to gastric cancer is questionable. Furthermore, we herein investigated whether and how autophagy plays a role in MSI-relevant chemoresistance.A total of 929 patients with deficient MMR (dMMR) and proficient MMR (pMMR) gastric cancers who underwent curative-intent gastrectomy were enrolled. We compared clinicopathological variables and survival among dMMR and pMMR cohorts and tested the responses of MSI-high and microsatellite stable (MSS) gastric cancer cell lines to 5-fluorouracil (5-FU) with or without chloroquine, an autophagy inhibitor.We identified an 8.9% prevalence of dMMR cases (83 out of 929) in our cohort. This was associated with old age, tumor site at the distal stomach, an intestinal phenotype, fewer nodal metastasis, and early pathological stages. MMR was an independent prognostic factor after multivariate adjustment. Overall survival (OS) of dMMR patients was better than that of the pMMR patients but was only applicable to stage III patients. There was no difference in OS between dMMR patients treated with or without adjuvant chemotherapy, although the latter showed more medical morbidities. The MSI-high gastric cancer cell lines, versus the MSS counterparts, displayed increased resistance to 5-FU and increased autophagy. Interestingly, autophagy inhibition abrogated the chemoresistance.Our data show that fluorouracil-based adjuvant chemotherapy does not work for dMMR cases, if not worse. Autophagy inhibition and/or immune checkpoint inhibition might be promising alternative strategies for gastric cancer treatment.The use of microsatellite instability (MSI) and mismatch repair (MMR) as predictive biomarkers for adjuvant chemotherapy in colorectal cancer has caused a paradigm shift in cancer therapy, although its implications in gastric cancer are still questionable. The data obtained in the current study indicate that MSI-MMR is an independent prognostic factor for gastric cancer. Standard fluorouracil-based adjuvant chemotherapy did not work for deficient MMR cases, and was likely worse. Instead, strategies like autophagy inhibition and/or immune checkpoint inhibition should be taken into consideration in the future.