Hypsizigus marmoreus is an edible mushroom that is cultivated worldwide. In this study, we investigated antiinflammatory activities of H. marmoreus extract on atopic dermatitis (AD)-like symptoms. Ethanol extract of H. marmoreus (HMEE) was administrated in powder to BALB/c mice in which AD was induced by 1-chloro 2, 4, 6-trinitrobenzene [picryl-chloride, (PCL)]. The dermatitis severity score and the thickness of the epidermis were significantly decreased following daily intake of HMEE powder (1 g/kg/day) for 5 weeks compared with a PCL-treated group. The mRNA expression of proinflammatory cytokines, such as interleukin-1 beta (IL-1β) and interferon-gamma (IFN-γ), was significantly attenuated in the dorsal skin of the HMEE-fed mouse group compared with the PCL-treated mouse group. In addition, in concanavalin A-stimulated and lipopolysaccharide-stimulated mouse splenocytes and macrophages, levels of IL-1β and IFN-γ production were attenuated following the addition of HMEE. Interestingly, the administration of HMEE to mouse splenocytes stimulated the production of an antiinflammatory cytokine, IL-4. However, increases in the levels of proinflammatory cytokines and nitric oxide were attenuated by treating the mouse splenocytes, mouse macrophages, and Raw 264.7 cell line with HMEE. These results strongly suggest that HMEE exhibits anti-AD activity via the regulation of inflammatory responses.