The hepatoprotective activity of ethanol extract of Ganoderma lucidum (GLE) in alcohol-induced liver injury in mice was investigated. HPLC coupled with photo-diode array detector and electrospray ionization-mass spectrometry was used to analyze the major triterpenoids in GLE. The effects of GLE on hepatoprotection were evaluated through histopathology and biochemical analysis of serum enzymes. We used isobaric tag for relative and absolute quantitation (iTRAQ) coupled with tandem mass spectrometry to identify differentially expressed liver proteome in mice. There were more than 4000 differentially expressed proteins; 40 proteins with the most significant changed proteins were applied for further bioinformatics analysis. Expression levels of cytochrome P450 2E1 and alcohol dehydrogenase 1, proteins that are closely associated with these processes, were validated by western blotting. Triterpenoids, major components of GLE, protected alcohol-induced liver injury through inhibiting lipid peroxidation, elevating activity of antioxidant enzymes, and suppressing apoptotic cell death and immune inflammatory response.