Poria cocos is an important Oriental medical fungus with multiple functionalities, yet its bioactive substances and the mechanisms involved have not been fully characterized. A novel immunomodulatory protein (P. cocos immunomodulatory protein; PCP) was purified from the dried sclerotium of P. cocos (Schw.) Wolf using DE-52 cellulose and gel filtration chromatography. Chromatography and electrophoresis results indicated that the native PCP (35.6 kDa) is a disulfide-linked heterodimeric glycoprotein consisting of 14.3 and 21.3 kDa subunits with N- and O-glycosylation. PCP was capable of stimulating RAW 264.7 macrophages in vitro through the induction of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1beta) as well as the regulation of nuclear factor-kappa B (NF-kappaB)-related gene expression. In primary mouse macrophages, PCP directly activated peritoneal cavity macrophages to induce Toll-like receptor 4 (TLR4)-mediated myeloid differentiation factor 88 (MyD88)-dependent signaling. This study demonstrated the cell surface interactions of PCP with TLR4 and the capacity of PCP for TLR4 tyrosine phosphorylation. Results obtained with peritoneal macrophages from TLR4-deficient C57BL/10ScN mice revealed that PCP-induced activation and PCP cell surface binding were significantly attenuated. Moreover, enzymatic deglycosylation decreased PCP-mediated responses, indicating that the glycosylated portion of PCP was a key factor in PCP signaling through TLR4 in peritoneal macrophages. These findings suggest that PCP is a new potential immune stimulator within P. cocos and that TLR4 is primarily responsible for PCP signaling in murine macrophages.