Glioma malignancy greatly depends on its aggressive invasion. The establishment of cell polarity is an important initial step for cell migration, which is essential for cell-directional translocation. However, our understanding of the molecular mechanisms underlying cell polarity formation in glioma cell invasion remains limited. Glycogen synthase kinase-3 (GSK-3) has a critical role in the formation of cell polarity. We therefore investigated whether localized GSK-3β, a subtype of GSK-3, is important for glioma cell invasion. We reported here that the localized phosphorylation of GSK-3β at the Ser9 (pSer9-GSK-3β) was critical for glioma cell invasion. Scratching glioma cell monolayer up-regulated pSer9-GSK-3β specifically at the wound edge. Inhibition of GSK-3 impaired the cell polarity and reduced the directional persistence of cell migration. Consistently, down-regulation of GSK-3α and 3β by specific small interfering RNAs inhibited glioma cell invasion. Over-expressing wild-type or constitutively active forms of GSK-3β also inhibited the cell invasion. These results indicated the polarized localization of GSK-3 regulation in cell migration might be also important for glioma cell migration. Further, EGF regulated both GSK-3α and 3β, but only pSer9-GSK-3β was enriched at the leading edge of scratched glioma cells. Up- or down-regulation of GSK-3β inhibited EGF-stimulated cell invasion. Moreover, EGF specifically regulated GSK-3β, but not GSK-3α, through atypical PKC pathways. Our results indicated that GSK-3 was important for glioma cell invasion and localized inhibition of GSK-3β was critical for cell polarity formation.