Regulatory macrophages (Mregs) are unique in that they have anti-inflammatory and immunosuppressive properties. Thus, treating inflammatory diseases using Mregs is an area of active research. Human Mregs are usually generated by culturing peripheral blood monocytes stimulated using a macrophage colony-stimulating factor with interferon (IFN)-γ. Herein, we generated Mregs with an elongated cell morphology from THP-1 cells that were stimulated with phorbol 12-myristate 13-acetate and cultured with both arginylglycylaspartic acid and vitamin D3. These Mregs regulated macrophage function, and respectively downregulated and upregulated the expression of pro-inflammatory and immunosuppressive mediators. They also expressed Mregs-specific markers, such as dehydrogenase/reductase 9, even when exposed to such inflammatory stimulants as IFN-γ, lipopolysaccharide, purified xenogeneic antigen, and xenogeneic cells. The Mregs also exerted anti-inflammatory and anticoagulatory actions in response to xenogeneic cells, as well as exerting immunosuppressive effects on mitogen-induced Jurkat T-cell proliferation. Our method of generating functional Mregs in vitro without cytokines is simple and cost-effective.