Objective: CD4+CD25+Foxp3+ regulatory T (Treg) cell-mediated immunosuppression is an essential mechanism of rheumatoid arthritis (RA). However, little is known regarding the specific role of CD4+CD25−Foxp3+ Treg cells in RA. This study aimed to investigate the frequency of circulating CD4+CD25−Foxp3+ Treg cells and their role in RA. Methods: Sixty-one untreated RA patients and 40 healthy controls (HCs) were enrolled in this study. The proportion of CD4+CD25−Foxp3+ T cells and CD4+CD25+Foxp3+ Tregs; the levels of CTLA4, GITR, Helios, and ICOS; and the production of IL-17A, IFN-γ, and IL-10 were assessed by flow cytometry. The correlation of CD4+CD25–Foxp3+ T cells and CD4+CD25+Foxp3+ Tregs with the clinical indicators was conducted by Spearman correlation analysis. Results: The proportion of CD4+CD25–Foxp3+ T cells was elevated in RA and positively correlated with disease activity. CD4+CD25–Foxp3+ T cells expressed less Helios and produced more IFN-γ than conventional Tregs in RA. Additionally, the proportion of CD4+CD25–Foxp3+ T cells was positively correlated with DAS28 score, IgG titer, and anti-CCP titer. Conclusions: These data indicate that CD4+CD25−Foxp3+ T cells in RA exhibit several different functional properties from conventional Tregs and are correlated with RA disease activity. Highlights: The proportion of CD4+CD25−Foxp3+ T cells was elevated in untreated RA. CD4+CD25−Foxp3+ T cells expressed decreased Helios compared with Treg cells in both RA patients and HCs. CD4+CD25−Foxp3+ T cells produced increased IFN-γ compared with Tregs in RA patients. Elevated CD4+CD25−Foxp3+ T cells were positively correlated with DAS28 score, anti-IgG titer and anti-CCP titer. [ABSTRACT FROM AUTHOR]