The immune regulatory function of macrophages (M⊘s) in mixed chimeras has not been determined. In the present study, with a multi‐lineage B6‐to‐BALB/c mixed chimeric model, we examined the ability of donor‐derived splenic M⊘s in the induction of regulatory T cells (Treg). B6 splenic M⊘s from mixed chimeras induced significantly less cell proliferation, more IL‐10 and TGF‐β, and less IL‐2 and IFN‐γ productions of CD4⁺ T cells from BALB/c mice than naive B6 M⊘s did, whereas they showed similar stimulatory activity to the third part C3H CD4⁺ T cells. Importantly, highly purified donor F4/80⁺CD11c⁻ M⊘s efficiently induced recipient CD4⁺Foxp3⁺ Treg cells from CD4⁺CD25⁻Foxp3⁻ T cells. Furthermore, donor M⊘s of mixed chimeras produced more IL‐10 and less IFN‐γ than those of naive mice when cultured with BALB/c but not the third party C3H CD4⁺ T cells. Induction of recipient CD4⁺ Treg cells by donor M⊘s was significantly blocked by anti‐IL‐10, but not by anti‐TGF‐β mAb. Therefore, donor M⊘s have the ability to induce recipient CD4⁺Foxp3⁺ Treg cells in a donor antigen‐specific manner, at least partially, via an IL‐10‐dependent pathway. This study for the first time showed that, in mixed allogeneic chimeras, donor M⊘s could be specifically tolerant to recipients and gained the ability to induce recipient but not the third party Foxp3⁺ Treg cells. Whether this approach is involved in transplant immune tolerance needs to be determined.