Sex-specific differences exist in innate and adaptive immune responses and are mediated by hormone signaling. Estrogen is able to differentially modulate the development and differentiation of immune cells, including T cells. However, the effect of estrogen on T cell function, especially at concentrations other than physiological, remains controversial and incompletely understood. Immunotherapy is one of the most promising cancer treatments to date with a high probability of future enhancements. The adoptive transfer of genetically modified T cells can mediate tumor regression but there are still many hurdles to enhancing the proficiency of this treatment. This study demonstrates for the first time that one major aspect to consider for designing potent immunotherapies for cancer is the impact of the patient's sex. Herein, using two different Ag-specific T cell groups, we investigated the effect of sex and estrogen in antitumor effector responses, T helper cytokine secretion, and, importantly, on T cell whole polyfunctionality important for memory T cell development and survival. Major differences were observed in T cell function and polyfunctionality between sexes and on E2 treatment. The findings of this study may be critical to understand the results of immunotherapy on different patients and for the enhancement of immunotherapy for cancer.