Objective To investigate the reasons of HOXA5 overexpression in GBM and the molecular mechanism of miR-128-3p regulating the proliferation, invasion and apoptosis of glioblastoma multiforme. Methods After increasing and decreasing miR-128-3p expression in U87 cell lines by lentivirus transfection, the changes of HOXA5 expression were detected by Western blot, to explore the correlation between miR-128-3p and HOXA5 in GBM. The dual-luciferase reporter tests were performed to detect the target interaction of miR-128-3p with HOXA5. Through CCK-8 test, Transwell test, flow cytometric assay and tumor cell xenograft in nude mice, we verified molecular mechanism of miR-128-3p regulating the proliferation, invasion and apoptosis of GBM in vitro and in vivo. Results The expression level of HOXA5 was decreased in U87 cell line after miR-128-3p upregulation. In addition, the expression level of HOXA5 was increased in U87 cell line after miR-128-3p downregulation (P < 0.05). The expression level of HOXA5 was correlated negatively with the expression of miR-128-3p in U87 cell lines. MiR-128-3p targetedly interacted with 3'UTR of HOXA5 and inhibited the expression of HOXA5. The proliferation, invasion and anti-apoptosis of U87 cells were significantly decreased in the miR-128-3p+control group. Conclusion MiR-128-3p regulates negatively the proliferation, invasion and anti-apoptosis of GBM cells by targeting HOXA5. The overexpression of HOXA5 is induced by downregulation of miR-128-3p in GBM.