Glioblastoma multiforme (GBM) is the most common and most malignant adult brain tumor. A characteristic of GBM is their highly invasive nature, making complete surgical resection impossible. The most common gain-of-function alteration in GBM is amplification, overexpression, and mutations of the epidermal growth factor receptor (EGFR). The constitutively activated mutant EGFR variant III (EGFRvIII), found in approximately 20% of GBM, confers proliferative and invasive advantage. The signaling cascades downstream of aberrant EGFR activation contributing to the invasive phenotype are not completely understood. Here, we show myristoylated alanine-rich protein kinase C substrate (MARCKS), previously implicated in cell adhesion and motility, contributes to EGFR-mediated invasion of human GBM cells. EGFRvIII-expressing or EGF-stimulated human GBM cells increased expression, phosphorylation, and cytosolic translocation of MARCKS in a protein kinase C-alpha-dependent manner. Down-regulation of MARCKS expression with small interfering RNA in GBM cells expressing EGFRvIII led to decreased cell adhesion, spreading, and invasion. Elucidation of mechanisms that promote EGFRvIII-mediated tumorigenesis in GBM, such as MARCKS, provides additional understanding and potential biological targets against this currently terminal human cancer.