Crk-like (CrkL) is an adapter protein that has crucial roles in cell proliferation, adhesion, and migration. However, the expression pattern and potential mechanism of CrkL protein in glioblastoma multiforme (GBM) have not been fully elucidated. To determine roles of CrkL in cell signaling, proliferation, and migration, small interfering RNAs and plasmids transfection were used to suppress or overexpress CrkL in U87 and U251; soft-agar assay and wound-healing assay were used to observe cell invasiveness, migration, and proliferation. Erk1/2, Smad2, and matrix metalloproteinase 9 (MMP9) were also analyzed by western blot. CrkL was expressed in U87 and U251 cell lines and can be activated by transforming growth factor-beta 1 (TGF-β1) in vitro; CrkL knockdown significantly suppressed the expression of phosph-ERK1/2 and MMP9 but enhanced phosph-Smad2 expression compared with control (p <0.001). Overexpression of CrkL against control upregulated phosph-ERK1/2 and MMP9 and, at the same time, downregulated phosph-Smad2 (p <0.01). On the other hand, CrkL knockdown could significantly affect U87 and U251 invasiveness (p <0.01) and wound closure (p <0.01) using soft-agar assay and wound-healing assay. These studies suggest that CrkL efficiently mediates cell proliferation, migration, and invasion induced by TGF-β pathway in glioblastoma. Furthermore, CrkL can be used as a potential and efficient therapeutic target of GBM and may also mediate other signaling pathway.