Agaricus brasiliensis has been demonstrated to have potent antitumor activity. The activity is postulated to act through mediation of the host immune system. We have reported that A. brasiliensis extract (ABE) inhibited compound 48/80 induced a systemic anaphylaxis-like reaction, ear swelling response, and passive cutaneous anaphylaxis-like reaction in mice. There is some recent information available on the mechanism of antiallergic effects resulting from oral administration of ABE. However, information regarding how ABE may activate macrophages through intestinal epithelial cells is still limited. To clarify the mechanism of macrophages activation by ABE, a gut in vitro model constructed of Caco-2 and RAW264.7 cells was applied. Treatment of ABE to the apical compartment resulted in significant increases in tumor necrosis factor (TNF)-a production in the basolateral compartment. Moreover, addition of catalase to the basolateral compartment before ABE treatment suppressed TNF-a production completely, but the addition of superoxide dismutase did not suppress this at all. These data suggest that ABE could potentiate hydrogen peroxide emissions from Caco-2 cells into the basolateral side and activate macrophages, which is important in the immune system.