Background: GBM is the most common and the most lethal brain tumors with the 5-year survival rate of ~4% for patients over age 55. Recently people are exploring the potential of chimeric antigen receptor T (CAR-T) cell therapy in glioblastoma, yet the clinical outcome is limited. It’s reported that NKG2DLs are wildly expressed in glioma stem-like cells, which supports NKG2D system might play an important role in GBM therapy. Here we used NKG2D as antigen binding domain to construct a second generation of CAR (KD-025) for GBM treatment. Methods: U251 cell line as well as GBM cancer patient samples were evaluated for NKG2DLs expression. The KD-025 CAR T cells showed antigen-specific stimulation by cytokine secretion and target cell lysis. U251 were used to establish in vivo subcutaneous and xenograft models in NSG mice. Mice received a single treatment of 10 million KD-025 CAR-T cells intravenously. The main organs of mice were examined by hematoxylin and eosin (HE) staining after different doses of KD-025 administration. Results: NKG2DLs were detected on U251 cells and most of screened glioma patient samples. The KD-025 expression was > 50% on the surface of T cells confirmed by flow cytometry. Co-incubation of KD-025 CAR with U251 cell specifically upregulates TNFa, IFN-γ, IL-10 and IL-2 cytokines and strongly lysis tumor cells even at low E:T ratio (50-60% at 1:1, 70% at 10:1). Strikingly, KD-025 CAR demonstrate very potent anti-tumor activity in vivo. All the tumor cells are gone 14 days after single treatment of KD-025 CAR T cells. Regarding to T cell persistence, the CAR-T cells are barely detectable 24 days after injection, which is comparable with CD19 CAR in our experiments as well as published data. No obvious pathological changes were found in the tested organs. Conclusions: Our work with the KD-025 CAR contributes to the growing body of research committed to discovering a novel therapy for GBM. NKG2D ligands are highly expressed on human GBM samples. NKG2D based CAR T cells KD-025 potently respond to GBM and eliminate tumor in a xenograft mouse model with no obvious safety issue. The results support future clinical trial of KD-025 CAR in patients with GBM, where the need for effective treatment is great.