MicroRNAs (miRs) act as oncogenes or tumor-suppressor genes, and regulate the proliferation, apoptosis, invasion, differentiation, angiogenesis and behavior of glioma stem cells, which are important in glioma development and recurrence. The present study was performed to investigate the impact of miR-218-5p on stem cell properties and invasive ability of the A2B5+CD133- human glioma stem cell subgroup. qRT-PCR was used to detect miR-218-5p expression in non-cancerous brain and human glioma tissues, human glioma cell lines and human glioma stem cell lines. Lentivirus vectors encoding miR-218-5p and anti-miR-218-5p were constructed and stably transfected into A2B5+CD133- SHG-139s cells. Neurosphere formation Cell Counting Kit-8 (CCK-8) and Transwell assays, immunofluorescence and qRT-PCR analyses were used to explore the role of miR-218-5p in SHG-139s cells. qRT-PCR analysis showed that miR-218-5p expression was lower in human glioma tissues and cells than in non-cancerous brain tissues and normal human astrocyte cells, and lower in A2B5+CD133- (SHG-139s) cells than in CD133+ (SU2 and U87s) cells. The CCK-8 assay demonstrated that the growth curve was significantly inhibited in the miR-218-5p-SHG-139s cells compared to the miR-control, blank and anti-miR‑218-5p groups. The neurosphere formation assay indicated that upregulation of miR‑218-5p expression inhibited SHG-139s neurosphere formation. Immunofluorescence staining and qRT-PCR showed that miR-218-5p reduced stem cell marker (A2B5, nestin, PLAGL2, ALDH1 and Sox2) expression compared with the controls; however, immunofluorescence staining analysis showed that upregulation of miR-218-5p expression led to no difference in CD133 expression. miR‑218-5p reduced SHG-139s cell invasiveness in the Transwell assay and reduced MMP9 expression as detected in qRT-PCR and immunofluorescence analyses. All differences were statistically significant. miR-218-5p expression was lower in human glioma tissues, cells and the A2B5+CD133- human glioma stem cell subgroup. miR-218-5p may be a tumor-suppressor gene in glioma that functions by upregulating miR-218-5p expression, which inhibits the stem cell properties and invasive properties of SHG-139s cells.