Dendritic cells (DCs) are powerful activators of primary and secondary immune responses and have promising activity as anticancer vaccines. However, various populations of immune cells, including natural killer cells, regulatory T cells and especially cytotoxic T lymphocytes (CTLs), can inhibit DC function through cytotoxic clearance. Spontaneous tumor-specific CTL responses are frequently observed in patients before immunotherapy, and it is unclear how such pre-existing responses may affect DC vaccines. We used an adoptive transfer model to show that DC vaccination fail to induce the expansion of pre-existing CTLs or increase their production of interferon γ (IFNγ). The expansion and effector differentiation of naïve host CD8(+) T cells was also suppressed in the presence of CTLs of the same specificity. Suppression was caused by the cytotoxic functions of the adoptively transferred CTLs, as perforin-deficient CTLs could respond to DC vaccination by expanding and increasing IFNγ production. Proliferation and effector differentiation of host CD8(+) T cells as well as resistance to tumor challenge were also significantly increased. Expression of perforin by antitumor CTLs was critical in regulating the survival of vaccine DCs, while FAS/FASL and TRAIL/DR5 had a significant, but comparatively smaller, effect. We conclude that perforin-expressing CTLs can suppress the activity of DC-based vaccines and prevent the expansion of naïve and memory CD8(+) T cells as well as antitumor immune responses. We suggest that, paradoxically, temporarily blocking the cytotoxic functions of CTLs at the time of DC vaccination should result in improved vaccine efficiency and enhanced antitumor immunity.