Objective. To reveal the expression profile of miRNA in glioma and the effects of microRNA-339-5p (miR-339-5p) on glioma. Methods. The glioma and normal tissues were randomly selected for miRNA gene chip detection and qRT-PCR verification. The U87 cells were separated into miR-NC, miR-339-5p mimic, and miR-339-5p suppressor group. Clonogenesis test, flow cell technique, Transwell, and cell scratch assay were utilized to verify the roles of miR-339-5p in cell proliferation, cell apoptosis, cell invasion, and cell migration. The epithelial-meso-transformation-associated proteins was verified by Western blot. Results. A total of 49 miRNAs (16 upregulated and 33 downregulated) were differentially expressed in glioma tissues, and miR-339-5p was the most downregulated. The clone number, invasion number, and healing rate of cells in miR-339-5p mimic group were decreased compared with miR-NC group (
P
<
0.05
); the clone quantity, invasion number, and healing rate of cells in miR-339-5p inhibitor group were increased compared with miR-NC group (
P
<
0.05
). The apoptosis rate of human glioma U87 cells in miR-339-5P mimic group was compared with miR-NC group (
P
<
0.05
); the apoptosis rate of human glioma U87 cells in miR-339-5p suppressor group decreased compared with miR-NC group (
P
<
0.05
). Compared with miR-NC group, the protein expression of Twist, Snsnail, N-cadherin, and Vimentin in miR-339-5p mimic group was considerably decreased, whereas E-cadherin was elevated (
P
<
0.05
). Compared with miR-NC group, the protein expression of Twist, Snsnail, N-cadherin, and Vimentin in miR-339-5p suppressor group was considerably increased, whereas E-cadherin was considerably decreased (
P
<
0.05
). Conclusion. Forty-nine glioma-related miRNAs were screened out, and miRNA expression was significantly different between glioma and normal tissues. The downregulated miR-339-5p in glioma can regulate the proliferative, apoptotic, invasive, and migratory abilities of glioma U87 cells and might suppress the occurrence and development of glioma.