To investigate the effect of high-mobility group box 1 (HMGB1) protein on the regulatory T cells (Tregs) in vitro and its potential regulating mechanism in mice. Splenic CD4(+)CD25(+) Tregs and CD4(+)CD25(-) T cells were isolated. The time-dependent and dose-dependent responses between HMGB1 stimulation and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and forkhead/winged helix transcription factor p3 (Foxp3) expressions were analyzed. The secretion of various cytokines in the cell suspensions and the proliferation of CD4(+)CD25(-) T cells were determined. HMGB1 was found to be able to markedly down-regulate the expressions of CTLA-4 and Foxp3, especially at 72 h group and in 1,000 ng/mL group (both P < 0.01). The expression of Foxp3 mRNA showed a similar tendency as Foxp3 protein (P < 0.05 or P < 0.01). The secretion of interleukin (IL)-10 from Tregs was decreased when the concentration of HMGB1 was increased. The suppressive activity of proliferation of CD4(+)CD25(-) T cells exceeded 90% when the ratio of Tregs to CD4(+)CD25(-) T cells was 1:1; meanwhile, the proliferation of CD4(+)CD25(-) T cells was enhanced when cultured with HMGB1-stimulated Tregs. In the culture of HMGB1-stimulated Tregs, IL-2 and interferon-γ levels were elevated; whereas IL-4 and IL-10 decreased in CD4(+)CD25(-) T cells after the increased concentration of HMGB1 in comparison with unstimulated-Treg group. HMGB1 stimulation can result in markedly down-regulatory expressions of CTLA-4 as well as in Foxp3 expression and secretion of IL-10 from splenic Tregs in mice. HMGB1 appears to be involved in modulating cell-mediated immunity by influencing proliferation of effector T cells, secretion of IL-2, and cell polarization.