CD4(+)CD25(+) regulatory T cells (Tregs) play an important role in maintaining host immune tolerance via regulation of the phenotype and function of the innate and adaptive immune cells. Whether allogeneic CD4(+)CD25(+) Tregs can regulate recipient mouse macrophages is unknown. The effect of allogeneic donor CD4(+)CD25(+) Tregs on recipient mouse resident F4/80(+)macrophages was investigated using a mouse model in which allogeneic donor CD4(+)CD25(+) Tregs were adoptively transferred into the peritoneal cavity of host NOD-scid mice. The phenotype and function of the recipient macrophages were then assayed. The peritoneal F4/80(+) macrophages in the recipient mice that received the allogeneic CD4(+)CD25(+) Tregs expressed significantly higher levels of CD23 and programmed cell death-ligand 1(PD-L1) and lower levels of CD80, CD86, CD40 and MHC II molecules compared to the mice that received either allogeneic CD4(+)CD25(-) T cells (Teffs) or no cells. The resident F4/80(+) macrophages of the recipient mice injected with the allogeneic donor CD4(+)CD25(+) Tregs displayed significantly increased phagocytosis of chicken red blood cells (cRBCs) and arginase activity together with increased IL-10 production, whereas these macrophages also showed decreased immunogenicity and nitric oxide (NO) production. Blocking arginase partially but significantly reversed the effects of CD4(+)CD25(+) Tregs with regard to the induction of the M2 macrophages in vivo. Therefore, the allogeneic donor CD4(+)CD25(+) Tregs can induce the M2 macrophages in recipient mice at least in part via an arginase pathway. We have provided in vivo evidence to support the unknown pathways by which allogeneic donor CD4(+)CD25(+) Tregs regulate innate immunity in recipient mice by promoting the differentiation of M2 macrophages.