We have reported that D-Fraction, a polysaccharide extracted from the edible maitake mushroom (Grifola frondosa), activates immunocompetent cells, thereby eliciting antitumor activity. To extend the application of D-Fraction as a nutritional supplement for healthy people as well as treatment for those with cancer, we investigated the effects of D-Fraction on the immune system in normal C3H/HeJ mice. Splenocytes from mice administered D-Fraction intraperitoneally for 17 consecutive days were cultured, and the culture supernatants were analyzed for nitric oxide (NO) and interleukin (IL)-12 production by antigen-presenting cells (APCs), including macrophages and dendritic cells, and also for the T helper (Th)-1 cytokine interferon (IFN)-gamma and the Th-2 cytokines IL-4 and IL-10. The level of IL-10 as well as those of NO and IFN-gamma were increased by D-Fraction as compared with the control, in which the serum immunoglobulin E level was increased. The results suggest that D-Fraction induced a Th-2 dominant response through the activation of macrophages, resulting in the enhancement of humoral immunity rather than cell-mediated immunity. Furthermore, an increase in the percentage ratio of CD69 and CD89 expression on major histocompatibility complex II(+) cells revealed activation of APCs 4 h after D-Fraction administration. These results indicate that D-Fraction enhances both the innate and adaptive arms of the immune response in normal mice. Therefore, its administration may enhance host defense against foreign pathogens and protect healthy individuals from infectious diseases.