Background. Z-100 is a hot-water extract of the human-type Mycobacterium tuberculosis strain Aoyama B. While Z-100's macrophage-mediated immunomodulatory effects have been reported, the mechanistic details have not been fully clarified. Here, we studied the immunomodulatory effects of Z-100 on mouse bone marrow-derived cells, human CD14[sup.+] cells, and skin. Methods. Mouse bone marrow-derived cells and CD14[sup.+] cells were cultured in the presence of granulocyte-macrophage colony-stimulating factor, differentiated into macrophage-like cells, and then stimulated with Z-100. Furthermore, since Z-100 is subcutaneously administered clinically, we injected Z-100 into mice and measured gene expression in the skin. Results. While Z-100 stimulation increased the production of interleukin- (IL-) 12p40 and IL-1β in mouse bone marrow-derived macrophages, levels of IL-1β were low. In contrast, TNF-α production did not increase. Meanwhile, stimulation of human CD14[sup.+] cells with Z-100 increased production of IL-12p40, TNF-α , and IL-1β . Because Z-100 appeared to have the most stable effect on IL-12p40, we examined the components of Z-100 that induce IL-12p40 production. We found that Z-100 contained peptidoglycan-like components. In addition, an siRNA study showed that Z-100 increased the production of IL-12p40 via nucleotide-binding oligomerization domain 2 (NOD2). Further, subcutaneous administration of Z-100 to mice significantly elevated expression of IL-12p40 and IL-1β and showed a trend towards increasing TNF-α in the skin. Conclusion. Z-100 induced the production of immunomodulatory cytokines from various types of macrophages and specifically increased IL-12p40 production through peptidoglycan-like components via NOD2.