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Bioactive components in Ganoderma lucidummainly include polysaccharides (PS-G) and immunomodulatory protein Ling Zhi-8 (LZ-8). These components may have diverse regulatory functions in the immune system. However, the PS-G preparations from different procedures still contained partial LZ-8 residue, indicating that the specific target and regulating function of PS-G and LZ-8 were not fully understood. In the present study, PS-G was subjected to 15% TCA for removing proteins and the LZ-8 detection using anti-LZ-8 monoclonal antibodies showed a remarkable 89.7% protein reduction of the deproteinized PS-G (dpPS-G). The Saccharomyces cerevisiaewhich expressed recombinant LZ-8 protein (rLZ-8) without glycosylation was generated and then compared with dpPS-G in the induction toward murine primary macrophage and T lymphocytic cells. The peritoneal macrophages from TLR4-deficient and wild type mice revealed that TLR4 was a putative receptor of dpPS-G, mediating the TNF-α, IL-1β and IL-12p70 cytokine production and CD86, MHC II expression on macrophages, while rLZ-8 enhanced the production of IL-1β, IL-12p70, CD86, and MHC II expression by another obscure route. rLZ-8-treated macrophages enhanced the release of IFN-γ and IL-2 by murine CD4+and CD8+T cells, whereas dpPS-G treatment did not enhance the release of IFN-γ and IL-2. Furthermore, although the direct rLZ-8-treatment conduced dramatic CD154, CD44 expression on CD3+T cells and increased IL-2, IFN-γ secretion on CD4+and CD8+T cells, the dpPS-G was incapable of priming CD3+, CD4+or CD8+T cells unitarily. Taken together, these results demonstrated that LZ-8 could activate murine macrophages and T lymphocytes but PS-G was merely the activator for macrophages, suggesting their diverse roles in activating the innate and adaptive immunity.