Dendritic cells (DC) play a critical role in both the expansion of natural regulatory T cells (nTreg) and conversion of induced Treg (iTreg) from their precursors. In the present study, we evaluated the potential of DC to generate Treg from total CD4+ population which contains both nTreg and the precursors, and found that allogeneic (allo-DC) but not syngeneic DC (syn-DC) could effectively generated Foxp3+ Treg from total CD4+ population in the absence of exogenous cytokines. Compared with freshly purified CD4+ T cells, allo-DC-stimulated CD4+ T cells showed increased percentage of CD4+CD25+Foxp3+ Treg by 5–7-folds while syn-DC-stimulated CD4+ T cells did not. Furthermore, we demonstrated that the significant amounts of endogenous IL-2 and TGF-β, at least partially, contributed to the expansion of nTreg and conversion of iTreg in this cocultural system, respectively. Importantly, similar to nTreg, these allo-DC-generated Treg were capable of suppressing T cell response in vitro. Thus, our research provides a novel and efficient strategy for generation of Treg from total CD4+ population.