OBJECTIVES: As the prognosis of relapsed/refractory (R/R) acute myeloid leukaemia (AML) remains poor, novel treatment strategies are urgently needed. Clinical trials have shown that chimeric antigen receptor (CAR)-T cells for AML are more challenging than those targeting CD19 in B-cell malignancies. We recently developed -modified ligand-based CAR-T cells that target CD116/CD131 complexes, also known as the GM-CSF receptor (GMR), for the treatment of juvenile myelomonocytic leukaemia. This study therefore aimed to develop a novel therapeutic method for R/R AML using GMR CAR-T cells. METHODS: To further improve the efficacy of the original GMR CAR-T cells, we have developed novel GMR CAR vectors incorporating a mutated GM-CSF for the antigen-binding domain and G4S spacer. All GMR CAR-T cells were generated using a -based gene transfer system. The anti-tumor effect of GMR CAR-T cells was tested in mouse AML xenograft models. RESULTS: Nearly 80% of the AML cells predominant in myelomonocytic leukaemia were found to express CD116. GMR CAR-T cells exhibited potent cytotoxic activities against CD116 AML cells . Furthermore, GMR CAR-T cells incorporating a G4S spacer significantly improved long-term and anti-tumor effects. By employing a mutated GM-CSF at residue 21 (E21K), the anti-tumor effects of GMR CAR-T cells were also improved especially in long-term settings. Although GMR CAR-T cells exerted cytotoxic effects on normal monocytes, their lethality on normal neutrophils, T cells, B cells and NK cells was minimal. CONCLUSIONS: GMR CAR-T cell therapy represents a promising strategy for CD116 R/R AML.