Malignant gliomas persist as a major disease of morbidity and mortality in adult. Differentiation therapy has emerged as a promising candidate modality. However, the mechanism related is unknown. Here, we show that glycogen synthase kinase-3beta (GSK-3beta) is highly expressed and activated during the cholera toxin-induced differentiation in sensitive C6 and U87-MG malignant glioma cells, whereas the GSK-3alpha activity remains stable. GSK-3beta inhibitors or small interfering RNA suppress the induced-differentiation in sensitive C6 cells. Conversely, overexpression of a constitutively active form of human GSK-3beta (pcDNA3-GSK-3beta-S9A) mutant in resistant U251 glioma cells restores their differentiation capabilities. In addition, GSK-3beta triggers cyclin D1 nuclear export and subsequent degradation, which is necessary for differentiation in C6 and U251 glioma cells. Analysis of human glioma tissues further revealed overexpression of active GSK-3beta. These findings suggest that GSK-3beta is a differentiation fate determinant, and shed new lights on the mechanism by which GSK-3beta regulates cyclin D1 degradation and cellular differentiation in gliomas.