Glioblastoma (GBM) is a highly malignant brain tumor characterized by invasion tendency. Macrophage infiltration is associated with GBM invasion, but the mechanisms remain unclear. Hypoxia is an outstanding characteristic of GBM tissue. Hypoxia microenvironment modulates the biological behaviors of both tumor cells and infiltrated immune cells, including macrophages. In the present study, we analyzed the effects of hypoxia and macrophages on invasion of GBM cells and its potential mechanisms. We found that both hypoxia and macrophage supernatant promoted GBM cells invasion and matrix metalloproteinase (MMP)-9 expression, and hypoxia modulated the invasive activity of GBM cells by upregulating their CCR5 expression. The supernatant of hypoxic macrophages also showed greater pro-invasion effect than normoxic macrophages through the elevated secretion of CCL4. Moreover, we found that interferon regulatory factor-8 (IRF-8) was possibly involved in hypoxia-modulated CCL4 expression of macrophages. Taken together, the present study found that macrophages promoted GBM invasion by the CCL4-CCR5 axis, and hypoxia enhanced the interaction between these two types of cells by upregulating both CCL4 and CCR5 expression, respectively. The results of the present study suggested that hypoxia would be a potential target for the development of immune therapies of GBM.