Zymosan activates phagocytes through the innate immune system and causes inflammatory responses in animals. Because of the complexity of the active substances included in Zymosan preparations, simplifying the active moiety actually responsible for innate immune recognition is needed. One way to remove possible active substances from commercially available Zymosan preparations is to wash then with pyrogen-free water to obtain a ZWIS (Zymosan water insoluble fraction), ethanol insoluble (EtIS), or chloroform/methanol insoluble (CMIS) preparation. The effects of various washed Zymosan preparations on nuclear factor (NF)-κB activation and binding to β-glucan recognition protein were examined. Significant NF-κB activation by Toll-like receptor (TLR) 2-expressing HEK293 cells and enhanced NF-κB activity via the co-expression of TLR2 and Dectin-1, a functional β-glucan receptor, was only observed in response to ZWIS. However, the ability of Zymosan preparations to bind Dectin-1 protein was not altered even after treatment with the organic solvents by which the TLR2-mediated NF-κB activity was abolished. Another NF-κB activation pathway involving CARD9/Bcl10 was triggered by these Zymosan preparations in the presence of Dectin-1. The results suggest that the β-glucan-dependent characteristics of Zymosan were not affected by the washing with chloroform/methanol or ethanol, and that TLR2-mediated activity was easily eliminated with these organic solvents. This treatment might be useful for distinguishing natural ligands for TLR2 and β-glucan receptors when studying the innate immune response to fungal macromolecules.