Glioma is a deadly tumor that accounts for the majority of malignancies of the brain and central nervous system. Currently, applying biomarkers and combining with clinical treatments in the diagnosis and prognosis of Glioma is the optimal trend. Among the effective biomarkers, microRNA has emerged as a novel, efficient biomarker. In this study, we used data from the Kyoto Encyclopedia of Genome and Gene Routes Database (KEGG) and bioinformatics tools for functional prediction and annotation of two Glioma-associated molecules (miR-6780a-5p and miR-6754-3p). The result indicated that 361 genes relating with two molecules miR-6780a-5p and miR-6754-3p. In which, miR-6780a-5p interacts with 198 genes and miR-6754-3p interacts with 166 genes. PPI analysis (protein-protein interaction analysis) and functional enrichment analysis revealed that target molecules of two miRNAs (miR-6780a-5p, miR-6754-3p) belong to cancer signaling pathways such as MAPK (Mitogen-activated protein kinase) and WNT (Wingless-related integration site). In addition, two miRNAs (miR-6780a-5p and miR-6754-3p) affect not only Glioma but also regulation in many other cancers such as pancreatic, thyroid, and endometrial cancer. Briefly, the result has clarified the role of (miR-6780a-5p, miR-6754-3p) in regulating signaling pathway of Glioma, thereby evaluating the interaction between miRNAs, target genes and target signaling pathways, and also supporting for the selection of potential biomarkers in Glioma diagnosis