Mycelia of the edible mushroom Lentinus edodes (shiitake) were cultivated in a solid medium, and two fractions were obtained by hot-water extraction (L.E.M.) and then ethanol extraction followed by Sephadex LH-20 column chromatography (ESMe). The L.E.M. and ESMe were then examined for their hepatoprotective effect on dimethylnitrosamine-injured mice. Both fractions decreased the blood aspartate aminotransferase and alanine aminotransferase levels, partially inhibited the overaccumulation of collagen fibrils, and suppressed the overexpression of genes for α-smooth muscle actin and/or heat-shock protein 47 in the mice. Both fractions also inhibited the morphologic change and proliferation of isolated rat hepatic stellate cells (HSCs), which play a central role in liver fibrosis, in a dose-dependent manner and without cytotoxicity. The direct interaction between the extracts and HSCs appears to be important for the hepatoprotective activity. Polyphenols contained in both fractions are considered to be potential candidates for expressing the hepatoprotective effects. The finding of antifibrotic activity in extracts from an edible mushroom is expected to be helpful in the development of hepatoprotective agents with few side effects.