Background <p>Peritoneal dissemination poses a critical challenge in advanced gastric cancer and leads to poor outcomes. Exosomes offer a novel platform for the delivery of tumor-suppressive microRNAs (miRNAs). MicroRNA-338-3p (miR-338-3p), a known tumor suppressor, may target tumor cells and enhance the resistance of normal cells to tumor progression. This study evaluated the therapeutic potential of exosomes loaded with miR-338-3p (Exo338) in preventing peritoneal dissemination of gastric cancer.</p> Methods <p>Exosomes loaded with miR-338-3p using Exo-Fect^™ were characterized for efficient miRNA loading and delivery. In vitro assays assessed the effect of miRNA on gastric cancer cell lines, MKN45 and HGC27, and also the effects of Exo338 on cell adhesion. Additionally, the effect of miRNA on normal mesothelial cells (MeT-5A) was evaluated for resistance to tumor cell adhesion. In vivo, a peritoneal dissemination model in nude mice was used to evaluate tumor burden after intraperitoneal Exo338 administration.</p> Results <p>Overexpression of miR-338-3p inhibited the proliferation and adhesion of gastric cancer cells in vitro. Exosomes derived from tumor cells showed a lower uptake efficiency, whereas those from MeT-5A cells were efficiently taken up by normal mesothelial cells. Exo338 reduced the ability of tumor cells to adhere to normal mesothelial cells. In vivo, Exo338 administration in a peritoneal dissemination model showed a trend toward a reduced tumor burden.</p> Conclusions <p>Exosome-mediated delivery of miR-338-3p offers a promising approach for gastric cancer therapy by reducing tumor adhesion to mesothelial cells. This strategy underscores the potential of exosome-based miRNA therapies for the treatment of advanced gastric cancer.</p>