In innate immunity, activated natural killer (NK) cells attack and damage pathogens such as bacteria and virus without restriction by the MHC antigen. NK cells activated by IL-12 have been reported to recognize and kill tumor cells in perforin-mediated apoptosis. We have reported that D-Fraction, a polysaccharide extracted from the maitake mushroom (Grifola frondosa), activates macrophages, dendritic cells, and T cells and inhibits the growth of tumor cells. However, the effects of D-Fraction on NK cell function in the innate immune response are not well known. In the present study, we administered D-Fraction to MM-46 mammary tumor-bearing C3H/HeJ mice intraperitoneally for 3 consecutive days and investigated its effects on the activation and cytotoxicity of NK cells. D-Fraction significantly enhanced the cytotoxicity against NK-sensitive YAC-1 cells and the expression of CD223 on NK cells. D-Fraction also increased the expression of CD86 on macrophages. In addition, the levels of IL-12 in the culture supernatant of whole spleen cells and in serum increased, compared with the control corresponding to an increase in expression of IL-12 receptor betaI on NK cells. These results suggest that D-Fraction enhances the cytotoxicity of NK cells through the production of IL-12 by macrophages activated by D-Fraction.