Gastric cancer (GC) is a highly prone malignant tumor, which has attracted wide attention. This study investigated the expression and clinical value of miR-499a-5p in GC. A total of 105 patients with GC were included in this study. Simultaneously, 55 patients with benign stomach disorders and 45 healthy subjects were enrolled as controls. Real-time quantitative polymerase chain reaction was used to determine the expression of miR-499a-5p. The receiver operating characteristic curve was used to assess the diagnostic value of miR-499a-5p in GC. Kaplan-Meier and logistic analyses were used to evaluate the association between miR-499a-5p and GC prognosis. The levels of miR-499a-5p are markedly downregulated in GC and have a high diagnostic value. miR-499a-5p is closely linked to pathological features of GC. Overexpression of miR-499a-5p inhibits GC cell growth, migration, and invasion. Furthermore, miR499a-5p is also related to GC and 5-year survival, and is a risk factor for GC death. The levels of miR-499a-5p were markedly downregulated in GC and related to GC pathological features. It has the potential to become a biomarker for the diagnosis of GC.