Although chronic infections with viruses such as HIV and hepatitis C virus have been associated with regulatory T cell (Treg)-mediated suppression of virus-specific CD8⁺ T-cell activity, no causal relationship between Tregs and chronic viral set points has been established. Using transgenic mice in which Tregs can be selectively ablated, we now show that transient depletion of Tregs during a chronic retroviral infection allows exhausted CD8⁺ T cells to regain antiviral functions, including secretion of cytokines, production of cytotoxic molecules, and virus-specific cytolytic activity. Furthermore, short-term Treg ablation resulted in long-term reductions in chronic virus loads. These results demonstrate that Treg-mediated immunosuppression can be a significant factor in the maintenance of chronic viral infections and that Treg-targeted immunotherapy could be a valuable component in therapeutic strategies to treat chronic infectious diseases.