T-cell immunoglobulin and mucin-domain containing-3 (TIM3), generally known as an immune checkpoint receptor, is expressed on leukemic stem and progenitor cells (LSPCs) in acute myeloid leukemia (AML), and has an active role in LSC self-renewal. Therefore, TIM3 has been suggested as a potential target for AML treatment. Hence, we explored the feasibility of targeting TIM3 with chimeric antigen receptor (CAR) T-cells. Despite the expression of TIM3 on activated T-cells, TIM3 CAR T-cells were successfully generated from different healthy individuals with excellent in vitro expansion without signs of fratricide and sustained central-memory phenotype with minimal expression of exhaustion-related markers, including complete loss of TIM3 expression. TIM3 loss also did not affect effector functions since TIM3 CAR T-cells efficiently lysed TIM3 leukemic cell lines, produced Th1-predominant cytokines, successfully inhibited the colony-forming of TIM3 AML-derived LSPCs, and showed excellent AML tumor control in xenogeneic mouse models. Notably, TIM3 CAR T-cells did not affect healthy hematopoietic progenitor cells and healthy mature hematopoietic cells that express TIM3 at moderate levels, suggesting an optimal therapeutic window for the treatment of AML.