Abstract: AIM To investigate the mechanism of the protective effect of bicyclol on acetaminophen induced hepatotoxicity in mice. METHODS ³¹P-MRS spectra in vivo were determined by using surface coil technique. The membrane fluidity of mitochondria and the activity of mitochondrial ATPase were also determined by spectrofluorophotometry and spectrophotometry methods. RESULTS The hepatotoxicity of acetaminophen is related to the lipid peroxidation and covalent binding to macromolecules, which leads to damage of mitochondrial function. Our results showed that the decrease of ATP/Pi and the elevation of PME/ATP in acetaminophen intoxicated mice were significantly inhibited by two doses of bicyclol (100, 200 mg·kg^-1) pretreatment, which indicate that bicyclol has significant protective effect on the decrease of liver ATP content induced by acetaminophen. Acetaminophen significantly inhibited the activity of mitochondrial ATPase by its cytotoxic metabolite NAPQI N-acetyl-p-benzoquinone, which has the potential to react with sulfhydryl groups or through sulfhydryl group oxidation. Our results showed that the reduction of mitochondrial fluidity as well as the inhibitory effect of mitochondrial ATPase induced by acetaminophen were also reduced by bicyclol. CONCLUSION The effect of bicyclol on acetaminophen induced liver injury maybe partly due to its protective effects on hepatic energy metabolism and mitochondria function.