Glioma is the most common primary malignant tumor of the central nervous system, which results in both a poor prognosis and outcome because of the aggressive progression of disease, growth and resistance to surgery, chemotherapy, and radiotherapy. MiR-140-5p is a small, non-coding single-stranded RNA molecule, which was previously studied in the settings of human tongue cancer, hepatocellular carcinoma, and colorectal cancer. However, detailed data that formally demonstrate the contribution of miR-140-5p to glioma development are missing. Similarly, relatively little is known about the relationship of miR-140-5p, vascular endothelial growth factor A, and matrix metalloproteinase-2 in glioma progression. In this study, we found that miR-140-5p expression was significantly decreased in glioma tissues and in the glioma cell-lines U87 and U251 as compared with non-cancerous brain tissues by quantitative real-time polymerase chain reaction. In addition, miR-140-5p inhibited glioma cell proliferation and invasion and promoted glioma cell apoptosis both in vivo and in vitro. Interestingly, while the expression levels of miR-140-5p were higher in glioma cells, the messenger RNA or protein expression levels of vascular endothelial growth factor A and matrix metalloproteinase-2 were lower in glioma cells as determined by quantitative real-time polymerase chain reaction, western blot assay, and immunohistochemistry. By contrast, downregulation in the expression levels of miR-140-5p augmented the messenger RNA and protein expression levels of both vascular endothelial growth factor A and matrix metalloproteinase-2. These findings suggested that miR-140-5p inhibited glioma proliferation and invasion by regulating the vascular endothelial growth factor A/matrix metalloproteinase-2 signaling pathway both in vitro and in vivo.