The efficacy of cancer immunotherapy is limited because of central and peripheral immune tolerance towards tumor-antigens. We propose a novel approach based on the fact that the immune system has not evolved tolerance towards adenoviruses (Ads) and that Ads have not evolved efficient mechanisms for immune-escape. The host-response to intratumoral Ad-vector injection in mice that were immunologically tolerant to neu-positive syngeneic mammary-cancer (MMC) was investigated. Intratumoral injection with replication-deficient, transgene-devoid Ad induced immune responses at two different anatomical sites: the tumor-draining lymph nodes and the tumor microenvironment. The lymph nodes supported the generation of both neu- and Ad-specific T effector cells, while inside the tumor microenvironment only Ad-specific T cells expanded. Importantly, Ad-specific T cells were anti-tumor-reactive despite the presence of active regulatory T cell-mediated immune tolerance inside MMC tumors and anti-tumor efficacy of Ad was increased by pre-immunization against Ad despite the production of Ad-neutralizing antibodies.