Lentinula edodes (Berk) Pegler, commonly known as Shiitake mushroom has been used as medicinal food in Asian countries, especially in China and Japan and is believed to possess strong immunomodulatory property. In the present study, the methanolic extract of the fruit bodies of L. edodes was investigated for cytoprotective effect against H2O2-induced cytotoxicity in human peripheral blood mononuclear cells (PBMCs) by measuring the activities of xanthine oxidase (XO) and glutathione peroxidase (GPx) . H2O2 at a concentration of 5 microM caused 50% inhibition of PBMCs viability. The extract improved the PBMC viability and exerted a dose-dependent protection against H2O2-induced cytotoxicity. At 100 microg/ml of extract concentration, the cell viability increased by 60% compared with the PBMCs incubated with H2O2 alone. The extract also inhibited XO activity in PBMC, while showing moderate stimulatory effect on GPx. However, in the presence of H2O2 alone, both the enzyme activities were increased significantly. The GPx activity increased, possibly in response to the increased availability of H2O2 in the cell. When the cells were pretreated with the extract and washed (to remove the extract) prior to the addition of H2O2, the GPx and XO activities as well as the cell viability were comparable to those when incubated with the extract alone. Thus, it is suggested that one of the possible mechanisms via which L. edodes methanolic extract confers protection against H2O2-induced oxidative stress in PBMC is by inhibiting the superoxide-producing XO and increasing GPx activity which could rapidly inactivate H2O2.