Tumor-associated antigens (TAAs) include overexpressed self-antigens (for example, Her2/neu) and tumor virus antigens (for example, HPV-16 E6/E7). Although in cancer patients, TAA-specific CD4+ and CD8+ cells are often present, they are not able to control tumor growth. In recent studies, it became apparent that tumor site-located immune evasion mechanisms contribute to this phenomenon and that regulatory T cells have a major role. We tested in Her2/neu breast cancer and HPV-16 E6/E7 cervical cancer mouse models, whether intratumoral expression of immunostimulatory proteins (ISPs), for example, recombinant antibodies (αCTLA-4, αCD137, αCD3), cyto/chemokines (IL-15, LIGHT, mda-7) and costimulatory ligands (CD80), through adenovirus(Ad)-mediated gene transfer would overcome resistance. In both the breast and cervical cancer model, none of the Ad.ISP vectors displayed a significant therapeutic effect when compared with an Ad vector that lacked a transgene (Ad.zero). However, the combination of Ad.ISP vectors with systemic T regulatory (Treg) depletion, using anti-CD25 mAb (breast cancer model) or low-dose cyclophosphamide (cervical cancer model) resulted in a significant delay of tumor growth in mice treated with Ad.αCTLA4. In the cervical cancer model, we also demonstrated the induction of a systemic antitumor immune response that was able to delay the growth of distant tumors. Ad.αCTLA4-mediated tumor-destructive immune responses involved NKT and CD8 T cells. In both models no autoimmune reactions were observed. This study shows that Ad.αCTLA4 in combination with systemic Treg depletion has potentials in the immunotherapy of cancer. © 2011 Nature America, Inc. All rights reserved.