Human glioblastoma is a malignant and aggressive primary human brain solid tumor characterized by severe hypoxia. Hypoxia can induce autophagy, which may result in chemoresistance and malignant progression of cancer cells. MicroRNAs (miRNAs) have been reported to modulate hypoxia‑induced autophagy in various types of cancers. In the present study, we observed that hypoxia‑inducible factor (HIF)‑1α expression was increased while miR‑224‑3p expression was decreased under hypoxia in a time‑dependent manner in glioma LN229 and astrocytoma U‑251MG cell lines, as deteced by western blot analysis and real‑time quantitative polymerase chain reaction. In addition, HIF‑1α knockout inhibited cell motility and chemosensitivity by negatively regulating the expression of miR‑224‑3p under a hypoxic condition by Transwell and MTT assay. Moreover, hypoxia increased the relative expression of ATG5 (autophagy‑related gene 5) and LC3 II/I with a decreased level of p62. These results were correlated with autophagy in a time‑dependent manner, suggesting that hypoxia induced autophagy in glioblastoma and astrocytoma cells. Through bioinformatic prediction and luciferase reporter assay, we confirmed that ATG5 is a target of miR‑224‑3p and ATG5 expression was negatively regulated by miR‑224‑3p. Knockdown of ATG5 inhibited cell mobility with increased chemosensitivity of glioblastoma cells under hypoxia. Moreover, overexpression of miR‑224‑3p also inhibited cell mobility with increased chemosensitivity of glioblastoma cells under hypoxia. However, activation of autophagy was able to counteract these effects of miR‑224‑3p. Furthermore, in vivo experiments indicated that the miR‑224‑3p mimic enhanced the chemosensitivity of LN229 cells to temozolomide by immunohistochemistry and TUNEL assay. In summary, our experiments elucidated that the HIF‑1α/miR‑224‑3p/ATG5 axis affects cell mobility and chemosensitivity by regulating hypoxia‑induced autophagy in glioblastoma and astrocytoma. Therefore, miR‑224‑3p could be a novel target against hypoxia‑induced autophagy in glioblastoma and astrocytoma.