A growing body of evidence has shown that professional antigen-presenting cells (APC) treated with transforming growth factor-β (TGF-β) can induce a systemic antigen (Ag)-specific tolerance, similar to anterior chamber-associated immune deviation (ACAID). However, the exact mechanism for immune tolerance induced by TGF-β-treated APC has not been elucidated. In this study, we showed that intravenous injection of ovalbumin (OVA)-pulsed APC treated with TGF-β2 induced a peripheral tolerance as evidenced by an impaired delayed-type hypersensitivity (DTH) response. CD4+ T cells from mice receiving an intravenous injection of TGF-β2-treated APC pulsed with OVA could adoptively transfer a specific tolerance to naïve mice. An increased frequency of FoxP3-expressing CD4+ CD25+ T cells was observed in mice with tolerance. CD4+ CD25+ T cells from TGF-β2-treated APC-injected mice produced a large amount of TGF-β1 and exhibited an in vitro antigen-specific suppressive activity. CD4+ CD25+ T cells from TGF-β2-treated APC-injected mice were able to inhibit the antigen-specific DTH response significantly when adoptively transferred to naïve mice. These results indicate that FoxP3-expressing CD4+ CD25+ T cells might be actively involved in the development of tolerance induced by TGF-β2-treated APC.