In hepatitis C virus (HCV) infection, the Th1-type immune response is involved in liver injury. A predominance of immunosuppressive regulatory T cells (Treg) is hypothesized in patients with persistently normal alanine aminotransferase (PNALT). Our aim was to clarify the role of Treg in the pathogenesis of PNALT. Fifteen chronically HCV-infected patients with PNALT, 21 with elevated ALT (CH) and 19 healthy subjects (HS) were enrolled. We determined naturally-occurring Treg (N-Treg) as CD4+CD25high+FOXP3+ T cells. The expression of FOXP3 and CTLA4 in CD4+CD25high+ cells was quantified by real-time reverse transcriptase-polymerase chain reaction. Bulk or CD25-depleted CD4+ T cells cultured with HCV-NS5 loaded dendritic cells were assayed for their proliferation and cytokine release. We examined CD127-CD25-FOXP3+ cells as distinct subsets other than CD25+ N-Treg. The frequencies of N-Treg in patients were significantly higher than those in HS. The FOXP3 and CTLA4 transcripts were higher in PNALT than those in CH. The depletion of CD25+ cells enhanced HCV-specific T cell responses, showing that co-existing CD25+ cells are suppressive. Such inhibitory capacity was more potent in PNALT. The frequency of CD4+CD127-CD25-FOXP3+ cells was higher in CH than those in PNALT. Treg are more abundant in HCV-infected patients, and their suppressor ability is more potent in patients with PNALT than in those with active hepatitis.