Interferon (IFN)-γ and interleukin (IL)-4 are considered to be important factors to regulate immune responses. Although the effects of IFN-γ or IL-4 on macrophage functions are well established, their cooperative action is not well documented. Inducible nitric oxide synthase (iNOS) or arginase (Arg)-1 is a representative marker of M1 or M2 macrophages and plays a role in the acceleration or suppression of inflammatory responses. At present, we examined the effect of simultaneous treatment with IFN-γ and IL-4 on macrophage expression of iNOS and Arg-1 in the murine macrophage cell line, RAW264.7. IFN-γ or IL-4 increased iNOS or Arg-1 protein production, respectively. Of note, IL-4 combined with IFN-γ synergistically increased Arg-1 protein production, whereas IL-4 inhibited IFN-γ-induced iNOS production. In addition, IL-4 combined with IFN-γ synergistically increased cell surface expression of programmed death ligand (PD-L) 2, which is involved in T cell suppression, whereas IL-4 completely inhibited IFN-γ-induced expression of CD86, which is responsible for T cell activation. It seems that macrophages highly expressing Arg-1 and PD-L2 may be induced in the presence of IFN-γ and IL-4 at the inflammatory site, and might contribute to regulate inflammatory immune responses.