Regulatory T (T _reg ) cells contribute to immune homeostasis but suppress immune responses to cancer. Strategies to disrupt T _reg cell–mediated cancer immunosuppression have been met with limited clinical success, but the underlying mechanisms for treatment failure are poorly understood. By modeling T _reg cell–targeted immunotherapy in mice, we find that CD4 ⁺ Foxp3 ⁻ conventional T (T _conv ) cells acquire suppressive function upon depletion of Foxp3 ⁺ T _reg cells, limiting therapeutic efficacy. Foxp3 ⁻ T _conv cells within tumors adopt a T _reg cell–like transcriptional profile upon ablation of T _reg cells and acquire the ability to suppress T cell activation and proliferation ex vivo. Suppressive activity is enriched among CD4 ⁺ T _conv cells marked by expression of C-C motif receptor 8 (CCR8), which are found in mouse and human tumors. Upon T _reg cell depletion, CCR8 ⁺ T _conv cells undergo systemic and intratumoral activation and expansion, and mediate IL-10–dependent suppression of antitumor immunity. Consequently, conditional deletion of Il10 within T cells augments antitumor immunity upon T _reg cell depletion in mice, and antibody blockade of IL-10 signaling synergizes with T _reg cell depletion to overcome treatment resistance. These findings reveal a secondary layer of immunosuppression by T _conv cells released upon therapeutic T _reg cell depletion and suggest that broader consideration of suppressive function within the T cell lineage is required for development of effective T _reg cell–targeted therapies.