Environmental factors affecting mycelial growth and exo-polysaccharide production from Pleurotus nebroden-sis Inzenga (PN) and biological activities of PN extracts in vitro were studied. The culture conditions for effective my-celial growth and exo-polysaccharide production were found to be 25oC, 5% of inoculum size, and an initial pH from6.5 to 7.0. When 5% of glucose was used, the maximum mycelial growth and exo-polysaccharide concentrations were8.3 and 3.07g/L, respectively. Among the various nitrogen sources, the mycelial growth and exo-polysaccharide pro-duction were very strong when polypeptone was used. In the case of the minerals sources, K2HPO4 and MgSO4·7H2Owere found to best support for mycelial growth and exo-polysaccharide production. Under optimal conditions and meth-ods, the maximum mycelial growth and exo-polysaccharide production were obtained after 10 days of culture, 12.84and 4.85g/L, respectively, in a jar fermentor. The effects of the PN extracts on the viability of three human cancer celllines and antioxidant activity were investigated in vitro. When cancer cells of the lung (A549), cervical region (HeLa)and colon (KM12C) were incubated at 6 mg/mL of the PN ethanol extracts, the viabilities of the HeLa and KM12Ccells were slightly decreased. However, the growth of the A549 cells was remarkably inhibited when the PN ethanolextract was over 4mg/mL. The antioxidant activity showed 46.2% at 40μL, which was about 3.2 fold higher thanthat of the PN methanol extract.