Isoorientin (ISO) is considered one of the most important flavonoid-like compounds responsible for health benefits, including the prevention of liver damage as well as antioxidant, anti-inflammatory, and anti-nociceptive activities. Our previous study showed that ISO inhibited the proliferation of hepatoma cells through increasing intracellular ROS levels. Interestingly, ISO protects rat liver cells against hydrogen peroxide-induced oxidation stress by decreasing intracellular ROS levels. Why are there different effects of ISO on ROS in different physiological and pathophysiological circumstances? The present study investigated the effect of ISO on mitochondrial respiratory chain complexes and phase II detoxifying enzyme activities in human hepatoblastoma cancer cells (HepG2), buffalo rat liver cells (BRL-3A) and human liver cancer cells (HL-7702). The results showed that intracellular ROS levels and the protein expression of the respiratory chain complexes was significantly (p<0.01) higher in the HepG2 cells than in the BRL-3A and HL-7702 cells. Additionally, ISO notably (p<0.01) increased ROS levels in the HepG2 cells, while no significance was found in the BRL-3A and HL-7702 cells. Furthermore, in the HepG2 cells, the protein expression of the respiratory chain complexes and the phase II detoxifying enzyme activities and GSH content were decreased by ISO (p<0.01), while ISO, in a certain range, enhanced the expression of the protein complexes and the phase II detoxifying enzyme activities and GSH content in BRL-3A and HL-7702 cells. All of these results demonstrated, for the first time, that ISO possesses a notable hepatoprotective effect, which might be mediated through the respiratory chain complexes and phase II detoxifying enzyme activities.