Regulatory T cells (T_reg cells) can activate multiple suppressive mechanisms in vitro after activation via the T cell antigen receptor, resulting in antigen-independent suppression. However, it remains unclear whether similar pathways operate in vivo. Here we found that antigen-specific T_reg cells activated by dendritic cells (DCs) pulsed with two antigens suppressed conventional naive T cells (T_naive cells) specific for both cognate antigens and non-cognate antigens in vitro but suppressed only T_naive cells specific for cognate antigen in vivo. Antigen-specific T_reg cells formed strong interactions with DCs, resulting in selective inhibition of the binding of T_naive cells to cognate antigen yet allowing bystander T_naive cell access. Strong binding resulted in the removal of the complex of cognate peptide and major histocompatibility complex class II (pMHCII) from the DC surface, reducing the capacity of DCs to present antigen. The enhanced binding of T_reg cells to DCs, coupled with their capacity to deplete pMHCII, represents a novel pathway for T_reg cell–mediated suppression and may be a mechanism by which T_reg cells maintain immune homeostasis.