Cathepsin L (CTSL) is a lysosomal cysteine protease overexpressed and secreted by tumor cells. Our previous study found that CTSL was involved in ionizing radiation (IR)-induced epithelial-mesenchymal transition (EMT) and the increase of glioma invasion and migration. However, the mechanisms by which CTSL promoted this IR-induced glioma migration and invasion remained unclear. In this study, we demonstrated that IR reduced glycogen synthase kinase-3β (GSK-3β) activity, via the CTSL-mediated phosphorylation of its serine-9 residue, in U251 cells. Moreover, inhibition of p-GSK-3βSer9 in overexpressing CTSL cells attenuated EMT and decreased the expression of snail, an EMT-related transcription factor. As a result U251 cell migration and invasion was inhibited compared to over-CTSL cells. Alternatively, when CTSL was activated by IR or exogenously overexpressed, CTSL promoted EMT by processing homeobox protein cut-like1 (CUX1) to produce the physiologically active p110 isoform. In brief, this study revealed that IR-induced EMT as well as migration and invasion of glioma cells are mediated by CTSL through the Akt/GSK-3β/snail and CUX1 pathways. Consequently, this research also led to the identification of a potential novel target for therapeutic intervention of glioma.