Objective. MiR-491-3p, as a tumor suppressor miRNA, was found decreased in many solid tissues. In this study, we aim to investigate miR-491-3p expression in gastric cancer with or without lymph node metastasis (LNM). Methods. GSE173215 dataset from Gene Expression Omnibus (GEO) was used to measure miRNA expression from tissue samples of gastric cancer patients. Moreover, gastric tumor tissues (non-LNM: n = 78; LNM: n = 68) were obtained to detect the miR-491-3p expression. Receiver operating characteristic (ROC) curve and Kaplan–Meier (KM) survival analysis, as well as Cox regression analysis, were performed to reveal the role of miR-491-3p in diagnosis and prognosis of gastric cancer. Results. According to GSE173215 datasets (t = −11.25, adjust
P
value = 1.30E-06) and our clinical results (0.390 ± 0.193 vs. 0.562 ± 0.166,
P
<
0.005
), the gastric cancer patients with LNM showed lower miR-491-3p expression than those without LNM, demonstrating a high diagnostic efficiency (sensitivity: 74.36%; specificity: 69.12%). In addition, both LNM and low miR-491-3p expression were correlated with the poor prognosis of gastric cancer. Furthermore, the LNM patient with low expression of miR-491-3p had the worse prognosis, but the non-LNM patient with high expression of miR-491-3p had the best prognosis. MiR-491-3p expression (HR = 0.003, 95%CI: 3.35E-04∼0.028) and LNM (HR = 2.326, 95%CI: 1.046∼5.173) were independent risk factors for gastric cancer. Conclusion. Downregulated miR-491-3p expression was found in gastric cancer, being a high diagnostic efficiency and an independent risk factor for gastric cancer, especially in those having LNM.