Baicalein (BA), one of the major bioactive flavonoids isolated from Scutellariae Radix, possesses various pharmacological activities. The present study aimed to investigate the protective effects of BA on tert-butyl hydroperoxide (t-BHP)-induced hepatotoxicity, and to investigate the potential mechanisms in LO2 cells. BA was demonstrated to possess protective properties against t-BHP injury in LO2 cells, as evidenced by MTT and lactate dehydrogenase assays. BA significantly prevented t-BHP-induced depolarization of mitochondrial membrane potential (MMP), decreased the percentage of apoptotic cells caused by t-BHP, and prevented intracellular reactive oxygen species (ROS) generation in LO2 cells. Furthermore, BA slightly triggered autophagy in LO2 cells, as evidenced by the elevation of LC3-II expression, while BA combined treatment with an autophagy inhibitor (chloroquine) or activator (rapamycin) did not alter the hepatoprotective properties. In conclusion, BA may possess a hepatoprotective effect against t-BHP-induced liver cell injury, dependent on ROS removal. Therefore, BA may represent a potential drug candidate in protecting hepatotoxicity.