Background and Aims Research has shown that microRNA-9 (miR-9) is downregulated in various malignant tumors, but its expression and function in gastric cancer remain unclear. In a previous study, we predicted using bioinformatics methods that Bcl-2-associated athanogene 4 (BAG4) might be a target gene of miR-9, and we also found that BAG4 is highly expressed in gastric cancer tissues. Therefore, this study investigated the expression and function of miR-9 in gastric cancer and its relationship with BAG4.Methods The expression levels of miR-9 in gastric cancer tissues and adjacent non-cancerous tissues, as well as in gastric cancer cell lines and normal gastric mucosal cells, were detected using qRT-PCR. After overexpressing and knocking down miR-9 in gastric cancer cells using miR-9 mimics and inhibitors, cell proliferation was assessed using the CCK-8 assay and colony formation assay, and cell invasion was evaluated using a Transwell invasion assay. The targeting relationship between miR-9 and BAG4 was analyzed using a luciferase reporter assay. Then, the expression of BAG4 in gastric cancer cells transfected with miR-9 mimics or si-BAG4 was detected by qRT-PCR and Western blot, and related functional experiments were performed for validation.Results The expression of miR-9 was significantly lower in gastric cancer tissues (vs. adjacent non-cancerous tissues) and gastric cancer cell lines (vs. normal gastric mucosal cells) (all P<0.05). The expression of miR-9 was significantly associated with tumor size, depth of tumor invasion, lymph node metastasis, distant metastasis, and TNM stage in gastric cancer patients (all P<0.05). Patients with high miR-9 expression had a significantly higher overall survival rate than those with low miR-9 expression (P=0.028). Overexpression of miR-9 in gastric cancer cells significantly reduced proliferation and invasion abilities, while miR-9 knockdown significantly enhanced these abilities (both P<0.05). The luciferase reporter assay indicated that BAG4 was a downstream target gene of miR-9. In gastric cancer cells, both mRNA and protein expression levels of BAG4 were significantly reduced after transfection with miR-9 mimics or si-BAG4, while both mRNA and protein expression levels of BAG4 were significantly increased after transfection with miR-9 inhibitors (all P<0.05). Proliferation and invasion abilities of gastric cancer cells transfected with si-BAG4 were significantly reduced, and the co-transfection of miR-9 inhibitors reversed the effects of si-BAG4 on cell proliferation and invasion (all P<0.05).Conclusion miR-9 is downregulated in gastric cancer, and its expression is closely related to adverse biological characteristics of gastric cancer. The mechanism of action of miR-9 may involve negatively regulating BAG4 expression, thereby affecting the proliferation and invasion of gastric cancer cells.