Thymosin beta 4 is a pleiotropic actin-sequestering polypeptide that is involved in wound healing and developmental processes. Thymosin beta 4 gene silencing promotes differentiation of neural stem cells whereas thymosin beta 4 overexpression initiates cortical folding of developing brain hemispheres. A role of thymosin beta 4 in malignant gliomas has not yet been investigated. We analysed thymosin beta 4 staining on tissue microarrays and performed interrogations of the REMBRANDT and the Cancer Genome Atlas databases. We investigated thymosin beta 4 expression in seven established glioma cell lines and seven glioma-initiating cell lines and induced or silenced thymosin beta 4 expression by lentiviral transduction in LNT-229, U87MG and GS-2 cells to study the effects of altered thymosin beta 4 expression on gene expression, growth, clonogenicity, migration, invasion, self-renewal and differentiation capacity in vitro, and tumorigenicity in vivo. Thymosin beta 4 expression increased with grade of malignancy in gliomas. Thymosin beta 4 gene silencing in LNT-229 and U87MG glioma cells inhibited migration and invasion, promoted starvation-induced cell death in vitro and enhanced survival of glioma-bearing mice. Thymosin beta 4 gene silencing in GS-2 cells inhibited self-renewal and promoted differentiation in vitro and decreased tumorigenicity in vivo. Gene expression analysis suggested a thymosin beta 4-dependent regulation of mesenchymal signature genes and modulation of TGFβ and p53 signalling networks. We conclude that thymosin beta 4 should be explored as a novel molecular target for anti-glioma therapy.