Fungal β-glucan, known to have immunostimulatory and antitumor activities, can be recognized by host immune cells as one of the pathogen-associated molecular patterns (PAMPs). Although there are several reports on the diverse immunostimulatory activities of β-glucan, little is known about the intracellular signal transduction of β-glucan. Stimulation of RAW264.7 macrophage cells with β-glucan from Ganoderma lucidum induced the expressions of dectin-1, toll-like receptor 2 (TLR2), TLR4, and TLR6 at the transcription stage. Treatment with β-glucan also induced inflammatory mediators such as macrophage inflammatory proteins (MIP)-1α, MIP-1β, MIP-1γ, interleukin (IL)-1β, and tumor necrosis factor (TNF)-α. Treatment of the cells with polymyxin B, an inhibitor of lipopolysaccharides (LPS), blocked the induction of inflammatory mediators in LPS- or β-glucan-stimulated systems. Pretreatment of the cells in our cell culture system with LY294002, a phosphoinositide 3-kinase (PI3K) inhibitor, or U0126, a mitogen-activated protein kinase/extracellular-signal-regulated kinase (MAPK/ERK) kinase (MEK)1/MEK2 inhibitor, led to a reduction in the induction of inflammatory mediators in a concentration-dependent manner. These results show that stimulation of the macrophage cells by β-glucan induced the expressions of both dectin-1 and TLRs. We also found that the PI3K/Akt and MEK pathways were involved in the induction of inflammatory mediators in macrophage cells during intracellular signal transduction of β-glucan.