Glioblastoma are among the most common forms of cancer affecting the central nervous system, and yet there is currently no effective means of treating them. In the current study, we reported that tousled-like kinase 2 (TLK2) is a key factor in glioblastoma that modulates SRC signaling, thereby driving tumor malignancy. TLK2 is commonly upregulated in glioblastoma, and such upregulation was associated with poor patient outcomes. TLK2 overexpression induced cell growth, migration, invasion, and epithelial-mesenchymal transition, and cell cycle arrest, while TLK2 knockdown had the opposite effect. SRC pathway inhibition by Saracatinib resulted in reduced TLK2-mediated glioblastoma migration, invasion, confirming a key role for SRC signaling in regulating the functions of TLK2. Together, our findings demonstrate that glioblastoma TLK2 overexpression acts as a key driver of tumor malignancy via SRC signaling pathway.