Abstract Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only curative option for relapsed or refractory acute myeloid leukemia (AML). However, more than half ultimately experience disease relapse that is associated with a dismal median survival of just 6 months, highlighting the need for novel therapies. In the current study we explore the therapeutic potential of targeting cyclin A1 (CCNA1), a cancer-testis antigen that is overexpressed in malignant blasts and leukemic stem cells. We demonstrate the immunogenicity of this antigen to native T cells, with >90% of donors screened mounting a specific response. The expanded cells were Th1 polarized, polyfunctional, and cytotoxic toward CCNA1+/HLA-matched tumor cell lines. Furthermore, these cells were exquisitely specific for CCNA1 and exhibited no reactivity against other cyclin family members, including CCNA2, which shares 56% homology with CCNA1 and is ubiquitously expressed in dividing cells. Lastly, the detection of CCNA1-specific T cells in AML patients post-HSCT was associated with prolonged disease remission, suggesting the protective potential of such endogenous cells. Taken together, our findings demonstrate the feasibility of targeting CCNA1 and the potential for therapeutic benefit associated with the adoptive transfer of reactive cells.