We have recently demonstrated that phosphorylation of SMAD specific E3 ubiquitin protein ligase 2 (Smurf2) at Thr249 (Smurf2^Thr249) plays an essential role for maintenance of stemness of the mesenchymal stem cells (Iezaki, Hiraiwa et al., Development 2018). Glioblastoma (GBM) is the most common high‐grade malignant glioma in adults. Emerging evidence indicates that glioma-initiating cells (GICs) contribute to drug resistance and tumor recurrence owing to their ability for self-renewal. Here, we show that phosphorylation of Smurf2^Thr249 plays an important role for maintenance of stemness of GICs and GBM progression. Smurf2^Thr249 phosphorylation was markedly decreased in GBM patients as well as in GBM model mice. Infection of Smurf2(T249A) mutant, in which the threonine was replaced with an alanine to prevent phosphorylation, significantly increased not only sphere formation ability of human GICs but tumor progression in a GBM mouse model. On the contrary, Smurf2 phospho-mimetic mutant (Smurf2(T249E)) decreased both of them. These findings highlight a critical role of Smurf2^Thr249 phosphorylation in maintenance of stemness of GICs and GBM tumorigenesis, thereby providing a novel target in GBM.