Background & Aims: CD4(+) regulatory T cells (Tregs) expand during chronic hepatitis C virus (HCV) infection, inhibit antiviral immunity and promote fibrosis. Direct-acting antiviral agents (DAA) have revolutionized HCV therapy. However, it is unclear if Tregs are normalized after DAA-induced HCV elimination. Methods: We analyzed Tregs before (baseline), at end of therapy (EOT), 12 and 24 weeks (SVR12, SVR24) and long-term (51 +/- 14 weeks) after EOT in 26 genotype-1-infected patients who were successfully treated with sofosbuvir (SOF) plus interferon (IFN)/ribavirin (n = 12) and IFN-free DAA regimens (SOF plus daclatasvir or simeprevir; n = 14). Frequency, phenotype and suppressor function of peripheral Foxp3(+) CD25(+) CD4(+) T cells were studied by multi-color flow cytometry and co-culture inhibition assays. Results: Frequencies and activation status of Foxp3(+) CD25(+) CD4(+) T cells remained elevated above those of normal controls in both treatment groups even long-term after HCV elimination. Coculture assays indicated a dose-response relationship for functional inhibition of autologous CD4(+) effector T cells and confirmed that activation of Tregs remained largely unchanged over the observation period. Unlike IFN-free regimens, SOF plus IFN/ribavirin induced a transiently increased frequency of Foxp3+ CD25(+) CD4(+) T cells at EOT (5.0% at baseline to 6.1% at EOT; p = 0.001). These Foxp3(+) CD25(+) CD4(+) T cells co-expressed the activation markers glycoprotein A repetitions predominant (GARP; p = 0.012) and tumor necrosis factor receptor superfamily, member 4 (OX-40; p = 0.001) but showed unchanged in vitro inhibitory activity. Conclusion: Although IFN-based DAA therapy induced transient expansion of activated Foxp3(+) CD25(+) CD4(+) T cells, neither IFN-based nor IFN-free DAA regimens normalized frequencies and activation status of Tregs one year after viral elimination. Persistence of immunosuppressive Tregs may thus contribute to complications of liver disease even long-term after HCV cure. Lay summary: In chronic hepatitis C virus (HCV) infection, CD4(+) regulatory T cells (Tregs) can reduce antiviral immune responses, promote liver fibrosis and may increase the risk for liver cancer, because they gradually expand during disease. Modern directacting antiviral agents (DAA) can "cure" hepatitis C in almost all treated patients. However, our study shows that DAA do not normalize the increased frequency and activation status of Tregs even long-term after HCV elimination. Tregs may persistently modulate functions of the immune system even after "cure" of hepatitis C. (C) 2016 European Association for the Study of the Liver. Published by Elsevier B. V. All rights reserved.