Background: Liver cancer is the fifth most common cancer and the third most common cause of cancer mortality worldwide with the 5-year survival rate of ~10%. In the past decade, some studies began to choose scFv as antigen binding domain for CAR-T cell therapy to treat liver cancer, yet the clinical outcome is limited so far. We developed a chimeric antigen receptor(CAR) targeting NKG2DLs (KD-025) to leverage adoptive T cell therapy as a powerful treatment modality for liver cancer. Methods: Liver cancer SMMC7721 cell line as well as liver 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. SMMC7721 solid tumors 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 mouse were examined by hematoxylin and eosin (HE) staining after different doses of KD-025 administration. Results: NKG2DLs were detected on SMMC7721 and most of screened liver cancer 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 SMMC7721 cell specifically upregulates TNFa, IFN-γ, IL-10 and IL-2 cytokines and strongly lysis tumor cells even at low E:T ratio (40% at 1:1, 60% at 10:1). Strikingly, KD-025 CAR demonstrated very potent anti-tumor activity in vivo. All the tumor cells are gone 14 days after single treatment of KD-025 CAR T cells. Two out of 6 mice displayed tumor recurrence on day 49. The rest 4 mice were tumor free till day 77 when the mice were scarified. Regarding the CAR-T cell persistence, KD-025 CAR-T cells are barely detectable 24 days after injection, which is comparable to CD19 CAR in the same experiment as well as reported data. No obvious pathological changes were found in the tested organs. Conclusions: NKG2D ligands are highly expressed on human liver cancer samples, but not normal tissues. NKG2D based CAR T cells KD-025 potently respond to hepatic tumors and eliminate liver tumor in a xenograft mouse model with no obvious safety issue. The results support future clinical trial of KD-025 CAR in patients with liver cancer.