Background: Gastric cancer is a malignant tumor with a high morbidity and mortality. MicroRNAs are important regulators of gene expression, influencing the progression of gastric cancer. This study aimed to reveal the role of microRNA-140 (miR-140) in gastric cancer cell proliferation and its potential mechanisms. Material/Methods: Gastric cancer tissues and cell lines BGC-823, SGC-7901, and HGC-27 were used to analyze miR-140 levels compared to normal tissues and cell line GES-1. In HGC-27 cells transfected with miR-140 mimic, we performed MTT, colony formation assay, and cell cycle assay by flow cytometry. SOX4, a predicted target of miR-140, was mutated to verify its regulation by miR-140, and was overexpressed to analyze its function in cell proliferation. Doxorubicin treatment was performed to investigate the effect of miR-140 on drug resistance. Results: miR-140 was down-regulated in gastric cancer tissues and cell lines, with the lowest expression level in HGC-27. miR-140 overexpression inhibited HGC-27 cell viability and colony formation and resulted in G0/G1 arrest. miR-140 suppressed SOX4 expression via binding to the 3' untranslated region, while the mutant SOX4 could not be regulated. Overexpressing SOX4 led to promoted cell viability, colony formation, and cell cycle progress. miR-140 overexpression also improved the anti-viability effects of doxorubicin, suggesting its potential in reducing the drug resistance of gastric cells. Conclusions: These findings suggest that miR-140 directly inhibits SOX4, which might be one of its mechanisms in suppressing gastric cancer cell proliferation. This study provides a promising therapeutic strategy for treating gastric cancer and facilitates microRNA research in various diseases.