Gene transfer of a factor VIII (FVIII) plasmid into hemophilia A (HemA) mice achieved supraphysiologic FVIII expression, but triggered production of high-titer FVIII-specific antibodies and loss of functional FVIII activity. To test whether FVIII-specific regulatory T cells (Tregs) can modulate immune responses against FVIII, we developed a HemA mouse model in which all T cells overexpressed Foxp3 (HemA/Foxp3-Tg). FVIII plasmid therapy did not induce antibody production in HemA/Foxp3-Tg mice. CD4(+)Foxp3(+) T cells isolated from plasmid-treated HemA/Foxp3-Tg mice significantly suppressed proliferation of FVIII-stimulated CD4(+) effector T cells. The percentage of CD4(+) T cells expressing CD25, glucocorticoid-induced tumor necrosis factor receptor, and cytotoxic T lymphocyte antigen 4 increased significantly in spleen and peripheral blood for 9 weeks. Mice receiving adoptively transferred Tregs from FVIII-exposed HemA/Foxp3-Tg mice produced significantly reduced antibody titers compared with controls after initial challenge with FVIII plasmid and second challenge 16 weeks after first plasmid treatment. Adoptively transferred Tregs engrafted and distributed at 2% to 4% in the Treg compartment of blood, lymph nodes, and spleens of the recipient mice and induced activation of endogenous Tregs; the engraftment decreased to negligible levels over 8 to 12 weeks. Antigen-specific Tregs can provide long-lasting protection against immune responses in vivo and limit recall responses induced by a second challenge via infectious tolerance.