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In this study, high pressure processing (HPP) was applied to extract polysaccharide from Lentinus edodes stems (LES) at ambient temperature. The effects of pressure,solvent-to-solid ratio and holding time on the yield of polysaccharide were studied by quadratic rotation-orthogonal composite experimental design. The linear effect of solvent-to-solid ratio was significant (p < 0.01). The interaction effect of pressure x holding time was significant (p < 0.05). The quadratic effects of pressure and solvent-to-solid ratio were significant (p < 0.05, p < 0.01, respectively). The optimal condition was: pressure at 210 MPa, solvent-to-solid ratio at 26 mL per 1g and holding time at 20 min, and the maximum yield of polysaccharide was 53.62 mg/g. Comparing to the conventional extraction method, HPP shows dominant advantages with higher yield and shorter extraction time. The use of HPP to extract polysaccharide from LES is a potential alternative to conventional methods.