Beta-glucan (BG) is a representative pathogen-associated molecular pattern (PAMP) produced by pathogenic fungi. SCG is a BG obtained from Sparassis crispa, which stimulates splenocytes in DBA/2 mice to produce cytokines, such as GM-CSF, IFN-γ, and TNF-α. In the present study, we analyzed the molecular mechanism of SCG-mediated cytokine synthesis using cytocharasin D (CytD), an inhibitor of actin polymerization. It was found that GM-CSF and TNF-α synthesis of splenocytes stimulated with SCG, but not with lipopolysaccharide, was significantly enhanced in the presence of CytD. CRDO, partially hydrolyzed linear 1,3-BG curdlan, stimulated splenocytes of DBA/2 mice slightly to produce cytokines. CRDO, acting as an antagonist in the presence of SCG, changed to a strong agonist in the presence of CytD. CytD also enhanced cytokine synthesis of bone marrow-derived dendritic cells. Taken together, cytokine productivity of BG was significantly dependent on molecular weight, and CytD treatment is useful to enhance the sensitivity for analyzing the immunostimulating activity of BG in vitro.