AbstractCombination therapy with anti‐cytotoxic T lymphocyte‐associated protein 4 (CTLA‐4) and anti‐programmed death‐1 (PD‐1) monoclonal antibodies (mAbs) has dramatically improved the prognosis of patients with multiple types of cancer, including renal cell carcinoma (RCC). However, more than half of RCC patients fail to respond to this therapy. Regulatory T cells (Treg cells) are a subset of highly immunosuppressive CD4+ T cells that promote the immune escape of tumors by suppressing effector T cells in the tumor microenvironment (TME) through various mechanisms. CTLA‐4 is constitutively expressed in Treg cells and is regarded as a key molecule for Treg‐cell‐mediated immunosuppressive functions, suppressing antigen‐presenting cells by binding to CD80/CD86. Reducing Treg cells in the TME with an anti‐CTLA‐4 mAb with antibody‐dependent cellular cytotoxicity (ADCC) activity is considered an essential mechanism to achieve tumor regression. In contrast, we demonstrated that CTLA‐4 blockade without ADCC activity enhanced CD28 costimulatory signaling pathways in Treg cells and promoted Treg‐cell proliferation in mouse models. CTLA‐4 blockade also augmented CTLA‐4‐independent immunosuppressive functions, including cytokine production, leading to insufficient antitumor effects. Similar results were also observed in human peripheral blood lymphocytes and tumor‐infiltrating lymphocytes from patients with RCC. Our findings highlight the importance of Treg‐cell depletion to achieve tumor regression in response to CTLA‐4 blockade therapies.