CD4⁺CD25⁺ regulatory T cells (CD25⁺ Tregs) play a key role in immune regulation. Since hepatitis C virus (HCV) persists with increased circulating CD4⁺CD25⁺ T cells and virus-specific effector T-cell dysfunction, we asked if CD4⁺CD25⁺ T cells in HCV-infected individuals are similar to natural Tregs in uninfected individuals and if they include HCV-specific Tregs using the specific Treg marker FoxP3 at the single-cell level. We report that HCV-infected patients display increased circulating FoxP3⁺ Tregs that are phenotypically and functionally indistinguishable from FoxP3⁺ Tregs in uninfected subjects. Furthermore, HCV-specific FoxP3⁺ Tregs were detected in HCV-seropositive persons with antigen-specific expansion, major histocompatibility complex class II/peptide tetramer binding affinity, and preferential suppression of HCV-specific CD8 T cells. Transforming growth factor β contributed to antigen-specific Treg expansion in vitro, suggesting that it may contribute to antigen-specific Treg expansion in vivo. Interestingly, FoxP3 expression was also detected in influenza virus-specific CD4 T cells. In conclusion, functionally active and virus-specific FoxP3⁺ Tregs are induced in HCV infection, thus providing targeted immune regulation in vivo. Detection of FoxP3 expression in non-HCV-specific CD4 T cells suggests that immune regulation through antigen-specific Treg induction extends beyond HCV.