The optimal culture condition of basidiomycete Sparassis crispa mycelia was determined for producing a large amount of antineoplastic constituent, i.e. β-1,3-glucan. Furthermore, a new extraction method with steam explosion as a pretreatment was attempted to increase the amount of antineoplastic constituent extracted from S. crispa mycelia. The shake-flask liquid culture of fungal mycelia could shorten a culture time up to about 1/3 compared with the conventional stationary liquid culture. The productivity of S. crispa mycelia reached its maximum at an initial glucose concentration of 30 g/L, a culture temperature of 25–30 °C, and pH 5. Steam explosion at a steam temperature of 225 °C for 5 min not only enhanced the amount of antineoplastic constituent extracted from the mycelia but also shortened the extraction time significantly.