The development of oral medications to help prevent liver injury is desirable, and some mushrooms contain chemicals that show promise as such a treatment. Here, we tested whether a hot-water extract (L.E.M.) of the cultured mycelia of an edible mushroom, Lentinus edodes, could protect primary cultured hepatocytes from D-galactosamine (GalN)-induced injury. GalN induced cell death in the hepatocytes, and this effect was completely suppressed by the addition of 0.5 mg/ml L.E.M. Polyphenolic compounds contained in the L.E.M. seemed to be responsible for the protective effect. We next examined the protective effect of L.E.M. in a GalN-induced liver injury model in rats. In rats that had been treated with L.E.M. given orally or intraperitoneally, GalN caused less leakage of aspartate aminotransferase and alanine aminotransferase, markers for liver injury, and a lower decrease in serum protein content, than in non-L.E.M.-treated rats. Histological analysis of the liver also showed a protective effect of L.E.M. Our findings indicate that L.E.M. administration is a promising treatment for protecting the liver from acute injury.