Glioblastoma multiforme (GBM) is an aggressive Grade IV astrocytoma with a poor survival rate. This is largely due to the GBM tumor cells migrating away from the primary tumor site along white matter tracts and blood vessels leading to secondary tumor sites. It is unknown whether the microenvironment nanotopography influences the biomechanical properties of the tumor cells. Although these tumor cells have an innate propensity to migrate, we believe that the nanotopography changes the biomechanical properties to enhance the migratory phenotype. To study this, we used an in vitro polycaprolactone aligned nanofiber film that mimics the nanotopography of the white matter tracts and blood vessels to investigate the mechanical properties of the GBM tumor cells. Our data demonstrate that the cytoskeletal stiffness, traction force, and focal adhesion area are inherently lower in invasive GBM tumor cells compared to healthy astrocytes. Moreover, the tumor cytoskeletal stiffness was significantly reduced when cultured on the aligned nanofiber films compared to smooth and randomly aligned nanofibers films. Analysis of gene expression also showed that tumor cells cultured on the aligned nanotopography upregulated key migratory genes and downregulated key proliferative genes. In addition, cell cycle analysis exhibited a reduced proliferative state on aligned nanofibers, highlighting the dichotomy between proliferation and migration observed in GBM. Finally, focal adhesions of tumor cells were larger and more elliptical when grown on the aligned fibers, suggesting a more migratory state. Therefore, our data demonstrate that the invasive potential is elevated when the tumor cells are cultured on an aligned nanotopography. This in vitro model can further be used to identify the GBM tumor cells’ response in a mimetic in vivo tumor microenvironment and elucidate how the aligned nanotopography transduces into altered gene and protein expression, thus providing a mechanism to target to inhibit the enhanced migratory behavior observed in these cells.