Accumulating evidence reveals a significant correlation between angiopoietin 2 (Ang2) expression and tumor invasion and metastasis in various human cancers, but the major focus of recent studies has been on the angiogenic effects of Ang2. We recently reported that Ang2-stimulated glioma cell invasion results from the up-regulation and activation of matrix metalloprotease 2 (MMP-2) in tumor cells. In this study, we identify a novel mechanism by which Ang2 stimulates MMP-2 expression leading to glioma cell invasion. We show that Ang2 interacts with α_vβ₁ integrin in Tie2-deficient human glioma cells, activating focal adhesion kinase (FAK), p130^Cas, extracellular signal–regulated protein kinase (ERK) 1/2, and c-jun NH₂-terminal kinase (JNK) and substantially enhancing MMP-2 expression and secretion. The Ang2/α_vβ₁ integrin signaling pathway was attenuated by functional inhibition of β₁ and α_v integrins, FAK, p130^Cas, ERK1/2, and JNK. Furthermore, expression of a negative regulator of FAK, FAK-related nonkinase, by U87MG/Ang2–expressing glioma xenografts suppressed Ang2-induced MMP-2 expression and glioma cell infiltration in the murine brain. These data establish a functional link between Ang2 interaction with α_vβ₁ integrin and glioma cell invasion through the FAK/p130^Cas/ERK1/2 and JNK-mediated signaling pathway. (Cancer Res 2006; 66(2): 775-83)