PD‐1 negatively regulates CD8⁺ cytotoxic T lymphocytes (CTL) cytotoxicity and anti‐tumor immunity. However, it is not fully understood how PD‐1 expression on CD8⁺ CTL is regulated during anti‐tumor immunotherapy. In this study, we have identified that the ADAP‐SKAP55 signaling module reduced CD8⁺ CTL cytotoxicity and enhanced PD‐1 expression in a Fyn‐, Ca²⁺‐, and NFATc1‐dependent manner. In DC vaccine‐based tumor prevention and therapeutic models, knockout of SKAP55 or ADAP showed a heightened protection from tumor formation or metastases in mice and reduced PD‐1 expression in CD8⁺ effector cells. Interestingly, CTLA‐4 levels and the percentages of tumor infiltrating CD4⁺Foxp3⁺ Tregs remained unchanged. Furthermore, adoptive transfer of SKAP55‐deficient or ADAP‐deficient CD8⁺ CTLs significantly blocked tumor growth and increased anti‐tumor immunity. Pretreatment of wild‐type CD8⁺ CTLs with the NFATc1 inhibitor CsA could also downregulate PD‐1 expression and enhance anti‐tumor therapeutic efficacy. Together, we propose that targeting the unrecognized ADAP‐SKAP55‐NFATc1‐PD‐1 pathway might increase efficacy of anti‐tumor immunotherapy.