OBJECTIVE: To investigate the functional interaction between miR-124-3p and integrin β1 (ITGB1) in gastric cancer. METHODS: A comprehensive approach integrating bioinformatic prediction with experimental validation was employed. The study used dual luciferase reporter assay, CCK-8 assay, RT-qPCR, western blotting, wound healing assay, and transwell assays to systematically investigate the role of the miR-124-3p/ITGB1 regulatory axis in gastric cancer cell models. RESULTS: This study identified a novel regulatory axis involving miR-124-3p and ITGB1. Mechanistic investigations demonstrated that miR-124-3p directly targets ITGB1, as confirmed by dual-luciferase reporter assays. Aberrant expression of miR-124-3p significantly suppressed ITGB1 mRNA and protein levels, leading to impaired oncogenic properties, including reduced proliferation, migration, and invasion of gastric cancer cells. Furthermore, ITGB1 downregulation inhibited gastric cancer cell growth and invasion while suppressing epithelial-mesenchymal transformation (EMT). Notably, key EMT regulators, such as E-cadherin, were up-regulated. These findings suggest that miR-124-3p-mediated ITGB1 downregulation effectively suppresses gastric cancer progression by inhibiting cell proliferation, invasion, and metastasis. CONCLUSIONS: miR-124-3p functions as a tumor-suppressive miRNA that inhibits gastric cancer development through targeting ITGB1. The miR-124-3p/ITGB1 axis provides novel insight and may serve as a promising biomarker for gastric cancer diagnosis and treatment.