Rationale:Glioblastoma (GBM) is an aggressive malignant primary brain cancer with poor survival. Hypoxia is a hallmark of GBM, which promotes tumor cells spreading (invasion) into the healthy brain tissue.Methods:To better elucidate the influence of hypoxia on GBM invasion, we proposed a data-driven modeling framework for predicting cellular hypoxia (CHPF) by integrating single cell transcriptome profiling and hypoxia gene signatures.Results:We characterized the hypoxia status landscape of GBM cells and observed that hypoxic cells were only present in the tumor core. Then, by investigating the cell-cell communication between immune cells and tumor cells, we discovered significant interaction between macrophages and tumor cells in hypoxic microenvironment. Notably, we dissected the functional heterogeneity of tumor cells and identified a hypoxic subpopulation that had highly invasive potential. By constructing cell status specific gene regulatory networks, we further identified 14 critical regulators of tumor invasion induced by hypoxic microenvironment. Finally, we confirmed that knocking down two critical regulatorsCEBPDandFOSL1could reduce the invasive ability of GBM under hypoxic conditions. Additionally, we revealed the therapeutic effect of Axitinib and Entinostat through the mice model.Conclusion:Our work revealed the critical regulators in hypoxic subpopulation with high invasive potential in GBM, which may have practical implications for clinical targeted-hypoxia cancer drug therapy.