Water-soluble protein-bound polysaccharides(β-D-glucan) extracted from the mycelia of Sparassis crispa belonging to basidomyces fungus was proved to be clinically important agent due to its strong immunostimulating activities. During the strain improvement program, it was found that cell mass increase was most important in order to enhance β-D-glucan productivity, since β-D-glucan amount was almost constant per unit cell mass. Therefore, attempts to enhance biomass amount were carried out in both shake flask and bioreactor cultures of Sparassis crispa mycelia. In the fermentation studies performed with multi-step bioreactor system consisting of two stages of growth-fermenter and one final stage of production-fermenter, the production level of β-D-glucan was found to considerably depend on the medium compositions of both growth and production cultures in the respective fermentation stage, not to mention the producers' morphology. Therefore, medium optimization process was performed using sequential statistical methods such as one factor at a time method(OFAT),  Plackett-Burman design, full or fractional factorial design(FFD), steepest ascent method(SAM) followed by response surface method(RSM). About 2-fold increase in the β-D-glucan production was observed in the optimized production medium, as compared to the β-D-glucan amount obtained by use of the previous medium.