To separate, purify and identify the acidic polysaccharide fraction in Boletus edulis, and to determine its influence on mouse lymphocyte proliferation. Crude polysaccharides were prepared from Boletus edulis by hot water extraction and ethanol precipitation. Acidic polysaccharide fractions were separated and purified from crude polysaccharides by DEAE-52 cellulose column, Sephadex G-100 and 0.1M NaCl solution elution. The relative molecular weight of the acidic polysaccharide fraction was determined by efficient liquid gel permeation method, the monosaccharide composition was analyzed by HPLC, and the influence of the acidic polysaccharide fraction on the activation and proliferation of mouse T and B cells was determined using the MTT assay. The purified acidic polysaccharide fraction isolated from Boletus edulis (APFB) with an average molecular weight (Mw) of 39668 was obtained. APFB was composed of eight monosaccharides, specifically mannose, xylose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and fucose. Wherein, galacturonic acid had the highest content (32.1%). Concentrations between 5 μg/ml and 640 μg/ml APFB promoted proliferation in vitro of mouse spleen T cells stimulated by ConA for 72 h (P <0.05). At APFB concentrations of 10 μg/ml and more, APFB significantly enhanced proliferation in vitro of mouse spleen B cells stimulated by LPS for 72 h (P <0.05). The acidic polysaccharide APFB separated and purified from Boletus edulis can enhance activation and proliferation in vitro of mouse spleen T and B cells. This suggests that APFB may be an effective immune regulator because it promotes effective humoral and cellular immune functions.