Glioblastoma multiforme (GBM) is the most malignant human primary brain tumor, and its infiltrative nature represents the leading cause for the failure of therapies and tumor recurrences. It is therefore crucial the knowledge of the molecular mechanisms underlying GBM invasion to identify novel therapeutic targets to limit motility. In this study, we evaluated the role of Epidermal growth factor receptor Pathway Substrate 8 (Eps8), a crucial regulator of the actin cytoskeleton dynamics accompanying cell motility and invasion, in GBM migration and invasiveness. We found that silencing of the protein by small interfering RNAs (siRNAs) abrogated the migratory and invasive capacity of three different human GBM cell lines both in 2-dimensional (2-D) and 3-dimensional (3-D) in vitro assays. The inhibitory effect on invasion was maintained independently by the migration mode utilized by the cells in our 3-D model, and was accompanied by an impaired formation of actin-based cytoskeletal protrusive structures. Our data propose Eps8 as a key molecule involved in the control of the intrinsic invasive behavior of GBM cells, and suggest that this protein might represent a useful target for the design of new drugs for the treatment of these tumors.