Different molecular subtypes of glioblastoma (GBM) have been recently identified, of which the mesenchymal subtype is associated with worst prognoses. Here, we report that transforming growth factor-β (TGF-β) is able to induce a mesenchymal phenotype in GBM that involves activation of SMAD2 and ZEB1, a known transcriptional inducer of mesenchymal transition in epithelial cancers. TGF-β exposure of established and newly generated GBM cell lines was associated with morphological changes, enhanced mesenchymal marker expression, migration and invasion in vitro and in an orthotopic mouse model. TGF-β-induced mesenchymal differentiation and invasive behavior was prevented by chemical inhibition of TGF-β signaling as well as small interfering RNA (siRNA)-dependent silencing of ZEB1. Furthermore, TGF-β-responding and -nonresponding GBM neurospheres were identified in vitro. Interestingly, nonresponding cells displayed already high levels of pSMAD2 and ZEB1 that could not be suppressed by inhibition of TGF-β signaling, suggesting the involvement of yet unknown mechanisms. These different GBM neurospheres formed invasive tumors in mice as well as revealed mesenchymal marker expression in immunohistochemical analyses. Moreover, we also detected distinct zones with overlapping pSMAD2, elevated ZEB1 and mesenchymal marker expression in GBM patient material, suggestive of the induction of local, microenvironment-dependent mesenchymal differentiation. Overall, our findings indicate that GBM cells can acquire mesenchymal features associated with enhanced invasive potential following stimulation by secretory cytokines, such as TGF-β. This property of GBM contributes to heterogeneity in this tumor type and may blur the boundaries between the proposed transcriptional subtypes. Targeting TGF-β or downstream targets like ZEB1 might be of potential benefit in reducing the invasive phenotype of GBM in a subpopulation of patients.