Glioblastoma is the most aggressive malignant primary brain tumor in humans, with extremely poor patient survival. Although previous studies have demonstrated that expression of matrix metalloproteinase-9 (MMP9) in glioblastoma promotes cancer metastasis, the upstream molecular signaling cascades that control activation of MMP9 remain largely unknown. Here, we used a human glioblastoma line, A-172, to examine molecular signaling to activate MMP9. We found that epidermal growth factor (EGF)-induced activation of epidermal growth factor receptor (EGFR) in A-172 cells activated MMP9, resulting in an increase in cancer invasiveness. A specific inhibitor for EGFR efficiently blocked EGF-induced activation of MMP9 and then cancer invasiveness. Moreover, an inhibitor for phosphatidylinositol 3-kinase (PI-3 K)/protein kinase B (Akt) significantly inhibited the EGF-induced activation of MMP9. Furthermore, nuclear exclusion of a major Akt downstream target, Forkhead box protein O1 (FoxO1), was induced by Akt activation, which could be inhibited by either an EGFR inhibitor or by PI-3 K/Akt inhibitor. An expression of a constitutive nuclear form of FoxO1 significantly inhibited MMP9 activation induced by EGF. Taken together, these findings suggest that EGF/EGFR signaling activates downstream PI-3 K/Akt to induce FoxO1 nuclear exclusion, which activates MMP9 to promote glioblastoma invasiveness. Thus, FoxO1 appears to be a novel therapeutic target for inhibiting metastasis of glioblastoma.