Sugarcane bagasse has been utilized byGanoderma lucidumin high value, but this also produces tons of spent substrate. For comprehensive exploitation of sugarcane bagasse, this study was to evaluate the potential of mycelia extract from spentGanodermasubstrate (MESGS). In vivo assays, MESGS could alleviate acute liver injury induced by carbon tetrachloride in male Institute of Cancer Research (ICR) mice without toxicity. Analysis revealed that MESGS could effectively relieve the histopathological lesions of liver tissue. There are significant tendencies to descend for the levels of aspartate aminotransferase and alanine aminotransferase with the increasing doses of MESGS. Similarly, plasma creatinine levels of mice treating with MESGS were restored to 11.2 similar to 13.24 mu mol/L which approached to 9.48 mu mol/L of the normal control group. MESGS could mitigate symptoms of liver index raising and splenomegaly. It could be known that after acute liver injury, the mice with MESGS -treating had less side effects on liver, kidney and spleen. Meanwhile, the decreased activity of superoxide dismutase and increased malonaldehyde level were inhibited by MESGS treatment. In vitro assays, MESGS also exhibited strong antioxidant in a dose dependent manner by radicals scavenging activity with 1,1-Diphenyl-2-picrylhydrazyl, hydroxyl and 3-ethylbenzthiazoline-6-sulfonic acid diammonium salt. Furthermore, MESGS is rich in polysaccharide (22.13%) and polyphenolics (2.02%). These findings demonstrated that the MESGS displayed certain hepatoprotective effect on the acute liver injury by inhibiting oxidative stress. Therefore, the MESGS could be beneficial for drug development against chemical injury. It provides research basis for further improving high value conversion of sugarcane bagasse. [GRAPHICS] .