This study was conducted to improve the useful components and biological activity of Lentinula edodes fermented by lactic acid bacteria (LAB). Three LAB strains (Lactobacillus brevis KCCM 11904, L. plantarum KCCM 354469, and L. fermentum KCCM 12116) were inoculated and used for L. edodes hot water extract (10%, 20%, 30%) fermentation. LAB fermentation of L. edodes hot water extracts decreased pH and thus were more acidic than non-fermented L. edodes hot water extract. β-glucan and ergothioneine contents were increased by L. edodes in a concentration-dependent manner. The ergothioneine and β-glucan contents were highest in fermented with 30% L. edodes hot water extract fermented by L. plantarum and L. brevis (40.48 mg/ 100 g and 13.94%, respectively). The hepato-protective effect of fermented L. edodes hot water extracts by the three LAB were tested using Sprague-Dawley rat primary hepatocytes. In primary hepatocytes obtained following liver injury induced by acetaminophen, fermented L. edodes hot water extracts by the three LAB showed protective effects, as evident by reduction of the aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase liver markers. The collective results indicate that the fermented L. edodes hot water extracts obtained using LAB are potentially valuable in preventing or treating liver disease.