NKG2D ligands (NKG2DL) are expressed on various tumor types and immunosuppressive cells within tumor microenvironments, providing suitable targets for cancer therapy. Various immune cells express NKG2D receptors, including natural killer (NK) cells and CD8 T cells. Interactions between NKG2DL and NKG2D receptors are essential for NK-cell elimination of osteosarcoma tumor-initiating cells. In this report, we used NKG2D-NKG2DL interactions to optimize an immunotherapeutic strategy against osteosarcoma. We evaluated and the safety and cytotoxic capacity against osteosarcoma cells of CD45RA memory T cells expressing an NKG2D-4-1BB-CD3z chimeric antigen receptor (CAR). CD45RA cells from healthy donors were transduced with NKG2D CARs containing 4-1BB and CD3z signaling domains. NKG2D CAR expression was analyzed by flow cytometry. cytotoxicity of NKG2D-CAR CD45RA T cells against osteosarcoma was evaluated by performing conventional 4-hour europium-TDA release assays. For the orthotopic model, 531MII YFP-luc osteosarcoma cells were used as targets in NOD-scid IL2Rg mice. Lentiviral transduction of NKG2D-4-1BB-CD3z markedly increased NKG2D surface expression in CD45RA cells. Genetic stability was preserved in transduced cells. , NKG2D-CAR memory T cells showed significantly increased cytolytic activity than untransduced cells against osteosarcoma cell lines, while preserving the integrity of healthy cells. NKG2D-CAR memory T cells had considerable antitumor activity in a mouse model of osteosarcoma, whereas untransduced T cells were ineffective. Our results demonstrate NKG2D-4-1BB-CD3z CAR-redirected memory T cells target NKG2DL-expressing osteosarcoma cells and and could be a promising immunotherapeutic approach for patients with osteosarcoma. .