To investigate the immunomodulatory effects of Bacillus subtilis (B. subtilis) (natto) B4 spores on murine macrophage, RAW 264.7 cells were cultured alone or with B subtilis (natto) B4 spores at 37°C for 12 hrs, then both cells and culture supernatants were collected for analyses. Exposure of RAW 264.7 cells to B. subtilis (natto) B4 spores had no significant effects on macrophage viability and amounts of extracellular lactate dehydrogenase (LDH). However, it remarkably increased the activities of acid phosphatase (ACP), lactate dehydrogenase (LDH) and inducible nitric oxide synthase (iNOS) in cells and the amounts of nitric oxide (NO) and cytokines (tumor necrosis factor-alpha, interferon-gamma, interleukin [IL]-1 beta, IL-6, IL-12, IL-10 and macrophage inflammatory protein-2) in culture supernatants. These results demonstrate that B. subtilis (natto) B4 spores are harmless to murine macrophages and can stimulate their activation through up-regulation of ACP and LDH activities and enhance their immune function by increasing iNOS activity and stimulating NO and cytokine production. The above findings suggest that B. subtilis (natto) B4 spores have immunomodulatory effects on macrophages.