BACKGROUND/AIM: Gastric cancer (GC) is the third-leading cause of cancer-related deaths worldwide; thus, novel diagnostic and therapeutic biomarkers are needed. Annexin A10 (ANXA10) is a calcium- and phospholipid-binding protein. As far as we are aware, there are no reports describing the detailed functions of ANXA10 in GC. Therefore, we investigated the downstream mRNA variation and the effects of ANXA10 on chemoresistance in GC cell lines. MATERIALS AND METHODS: ANXA10 knockout GC cell lines were generated, and we performed functional analyses, chemosensitivity drug testing, and microarray analyses. Additionally, immunohistochemistry for ANXA10 was performed on 40 patients with GC who had received 5-fluorouracil (5-FU)-based chemotherapy to compare their prognosis and clinicopathological factors. RESULTS: ANXA10 knockout GC cells showed significantly increased proliferation, invasion, and sensitivity to 5-FU. The overall survival of ANXA10-positive cases was considerably lower than that of ANXA10-negative cases in GC patients who received 5-FU-based chemotherapy. Microarray analysis revealed candidate pathways regulated by ANXA10 and claudin 1 (CLDN1), keratin 80 (KRT80), RANBP2-type and C3HC4-type zinc finger containing 1 (RBCK1), and solute carrier family 7 member 5 (SLC7A5) genes. CONCLUSION: ANXA10 knockout increased the susceptibility of GC cell lines to 5-FU; ANXA10 may be a predictive indicator for response to 5-FU treatment in GC cases. ANXA10 may be involved in the pathogenesis of GC, in collaboration with CLDN1, KRT80, RBCK1, and SLC7A5.