Rheumatoid arthritis (RA) is an autoimmune and inflammatory disease. Natural T regulatory (nTreg) cells, which constitutively express the CTLA-4 molecule, have an important role in the pathogenesis of autoimmune conditions. Although it has been reported that biological agents are able to modulate the levels or function of Treg lymphocytes, the possible effect of Abatacept (CTLA-4-Ig) therapy on these cells has not been studied in autoimmune conditions. We explored the effect of Abatacept therapy on Treg cells in patients with RA. The number of different subsets of Treg cells was analyzed by flow cytometry in the peripheral blood from 45 patients with RA that were (n = 30) or not (n = 15) under Abatacept therapy as well as in 20 healthy controls. The function of Treg cells was assessed by an assay of inhibition of lymphocyte proliferation. We found that Abatacept therapy was associated with a significant diminution in the levels of CD4+CD25(bright)Foxp3+, and CD4+CTLA-4+ nTreg cells. In contrast, the regulatory function of CD4+CD25+ lymphocytes was significantly enhanced after the administration of Abatacept. Our data suggest that CTLA-4-Ig exerts a complex and interesting effect on Treg cells in patients with RA.