The effects of anticancer chemotherapy on antigen-specific cytotoxic T lymphocytes (CTLs) are mostly unknown. We tested the effects of cytotoxic drugs such as 5-fluorouracil, gemcitabine, and oxaliplatin on the functional activity of antigen-specific CTL cultures derived from the peripheral blood mononuclear cells of human donors. We found that a biweekly drug-exposure of human HLA-A(*)02.01+ CTLs derived from bulk cultures led to completely different effects if occurring early (day second) or late (day thirteenth) after the in vitro stimulations with the cognate peptides. In the first case, there was a significant CTL inhibition, whereas in the second, there was a marked enhancement of the antigen-specific cytolytic activity. Results of immunocytofluorimetric studies and CTL/natural killer inhibition assays suggested that the latter effect could be related to a more selective drug-mediated inhibition of cohabitant T regulatory (reg) cells. These results were translated in an in vivo therapeutic mouse model where humanized HLA-A(*)02.01 transgenic mice inoculated with EL-4/humanized HLA-A(*)02.01 transgenic mice showed a prolonged survival and the greatest rate of cure when receiving a combined treatment with a thymidylate synthase-specific peptide vaccine and a multidrug chemotherapy regimen administered late after immunization. Tumor samples derived from this group of mice showed a reduced expression of the target thymidylate synthase antigen, a marked reduction of T(reg)s, and a noteworthy infiltration of C8+ T cells. These results may have clinical implications for the design of new translational anticancer regimens aimed at combining chemotherapy and immunotherapy.