Background and Aims The long non-coding RNA SOX2 overlapping transcript (SOX2-OT) is involved in the regulation of cancer cell cycle and proliferation. Bioinformatics analysis has revealed potential binding sites among miR-409-3p, SOX2-OT, and membrane binding protein annexin A2 (ANXA2). This study aims to investigate the expression and functional role of the SOX2-OT/miR-409-3p/ANXA2 axis in gastric cancer cells.Methods qRT-PCR was used to measure the expression levels of SOX2-OT, miR-409-3p, and ANXA2 mRNA in gastric cancer tissues and cell lines. Gastric cancer cells were transfected with SOX2-OT shRNA plasmid (sh-SOX2-OT), co-transfected with sh-SOX2-OT and miR-409-3p inhibitor, or co-transfected with sh-SOX2-OT and ANXA2 overexpression plasmid. The control groups included blank, shRNA-negative control, inhibitor-negative control, and overexpression plasmid-negative control. Expression levels of SOX2-OT, miR-409-3p, and ANXA2 mRNA, cell proliferation, migration/invasion, apoptosis, and protein expression of Ki-67, cleaved caspase-3, Bax, MMP-9, and ANXA2 were assessed. Dual-luciferase reporter assays were conducted to confirm the targeting relationships among miR-409-3p, SOX2-OT, and ANXA2. A xenograft tumor model in nude mice was used to evaluate the effect of SOX2-OT on gastric cancer tumor growth in vivo.Results SOX2-OT and ANXA2 expression levels were significantly upregulated, while miR-409-3p was downregulated in gastric cancer tissues (vs. adjacent non-cancerous tissues) and gastric cancer cell lines (vs. normal gastric epithelial cells) (all P<0.05). In gastric cancer cels, knockdown of SOX2-OT led to decreased expression of SOX2-OT and ANXA2 mRNA and increased expression of miR-409-3p (all P<0.05), and this was accompanied by reduced proliferation and migration/invasion abilities, and increased apoptosis (all P<0.05); protein levels of ANXA2, Ki-67, and MMP-9 were significantly decreased, whereas cleaved caspase-3 and Bax levels were significantly increased (all P<0.05). These effects were reversed by co-transfection with the miR-409-3p inhibitor or ANXA2 overexpression plasmid (all P<0.05). Dual-luciferase assays confirmed the direct targeting relationships among miR-409-3p, SOX2-OT, and ANXA2. In vivo, knockdown of SOX2-OT significantly inhibited tumor growth in nude mice, with reduced SOX2-OT and increased miR-409-3p expression, as well as decreased ANXA2 and Ki-67 protein positivity in xenograft tissues (all P<0.05).Conclusion SOX2-OT is upregulated in gastric cancer cells and may promote malignant behaviors by competitively binding miR-409-3p, thereby relieving its inhibition on ANXA2. The SOX2-OT/miR-409-3p/ANXA2 axis may represent a potential molecular target for gastric cancer therapy.