Plasmacytoid dendritic cells (PDCs) are key effectors in host innate immunity and orchestrate adaptive immune responses. CpG oligodeoxynucleotides (ODN) have potent immunostimulatory effects on PDCs through TLR9 recognition and signaling. Little is known about the effects of CpG ODN on human PDC-mediated T cell priming. Here we show that type B CpG ODN effectively promotes PDCs to prime allogeneic naive CD4+ CD25− T cells to differentiate into CD4+ CD25+ regulatory T (Treg) cells. The CD4+ CD25+ T cells induced by CpG ODN-activated PDCs express forkhead transcription factor 3 and produce IL-10, TGF-β, IFN-γ, and IL-6, but low IL-2 and IL-4. These CD4+ CD25+ T cells are hyporesponsive to secondary alloantigen stimulation and strongly inhibit proliferation of autologous or allogeneic naive CD4+ T cells in an Ag-nonspecific manner. CpG ODN-activated PDCs require direct contact with T cells to induce CD4+ CD25+ Treg cells. Interestingly, IL-10 and TGF-β were undetectable in the supernatants of CpG ODN-stimulated PDC cultures. Both CpG-A and CpG-C ODN-activated PDCs similarly induced the generation of CD4+ CD25+ Treg cells with strong immune suppressive function. This study demonstrates that TLR9 stimulation can promote PDC-mediated generation of CD4+ CD25+ Treg cells and suggests PDCs may play an important role in the maintenance of immunological tolerance.