Regulatory T cells (Tregs) play pivotal role in cancer-induced immunoediting. Increment of CD25high+FOXP3+ natural Tregs has been reported in patients with hepatocellular carcinoma (HCC); however, the involvement of other type of Tregs remain elusive. We aimed to clarify whether FOXP3− Tregs are increased and functionally suppressive or not in patients with HCC. We enrolled 184 hepatitis C-infected patients with chronic liver diseases or HCC, 57 healthy subjects and 27 HCC patients with other etiology. Distinct Treg subsets were phenotypically identified by the expression of CD4, CD25, CD127 and forkhead/winged helix transcription factor (FOXP3). Their gene profiles, frequency and suppressor functions against T cell proliferation were compared among the subjects. To examine the molecules involving in Treg differentiation, we cultured naive CD4+ T cells in the presence of HCC cells and dendritic cells. We determined two types of CD4+CD127− T cells with comparable regulatory ability; one is CD25high+ cells expressing FOXP3 (CD25high+FOXP3+ Tregs) and the other is CD25− cells without FOXP3− expression (CD25−FOXP3− cells). The peripheral or intrahepatic frequency of CD25−FOXP3− Tregs in HCC patients is higher than those in other groups, of which significance is more than CD25high+FOXP3+ cells. Of importance, CD25−FOXP3− Tregs, but not CD25high+FOXP3+ cells, dynamically change in patients accompanied by the ablation or the recurrence of HCC. CD25−FOXP3− T cells with CD127−IL-10+ phenotype are inducible in vitro from naive CD4+ T cells, in which programmed cell death 1 ligand 1, immunoglobulin-like transcript 4 and human leukocyte antigen G are involved.. In conclusion, CD25−FOXP3− Tregs with suppressive capacity are increased in patients with HCC, suggesting their distinct roles from CD25+FOXP3+ Tregs.