Background. It is known that the infusion of whole blood from donors (donor-specific transfusion) into recipients combined with anti-CD154 therapy can prolong allograft survival. It has generally been agreed that the effectiveness of anti-CD154 therapy is caused by the inactivation of alloreactive CD4+ and CD8+ effector T cells. The recent literature has implicated CD4+CD25+ regulatory T cells in the suppression of autoimmunity and graft rejection, and we therefore examined whether CD154 blockade is effective because of its blockade of inflammatory T-cell activation or because of a direct impact on the regulatory T cells. Methods. RAG-/- mice were adoptively transfused with CD4+ T cells or a subset of the population (CD4+CD25+ or CD4+CD25- T cells) alone or in combination with donor-specific transfusion and anti-CD154 and given an allo-skin transplant. The longevity of the transplant was determined over time. CD154 -/-CD4+ T cells were used to assess the importance of CD154 in graft rejection and acceptance. Results. CD154 blockade (or loss of CD154) on CD4+CD25+ regulatory T cells enhanced their immunosuppressive activities and was a contributing factor to anti-CD154 -induced immune suppression in vivo. In a model of allograft tolerance, suppression was elicited by antigen and anti-CD154 or antigen alone if the CD4+CD25+ regulatory T cells were deficient in CD154 expression. Conclusions. Neutralizing the function of CD154 on regulatory T cells upon antigen exposure induces heightened levels of suppressive activities and is likely a contributing factor to the long-lived therapeutic effects of anti-CD154 treatment.