Oxidative stress contributes to the initiation and progression of liver damage. SIRT3 is a member of nicotinamide adenine dinucleotide-dependent deacetylases that plays a key role in anti-oxidative defense and mitochondrial function in the liver. Honokiol is a natural lignan from the plants of Magnolia genus that exhibits potent anti-oxidative property. This study aims to evaluate the hepatoprotective potential of honokiol against oxidative injury in tert-butyl hydroperoxide (t-BHP)-injured AML12 hepatocytes in vitro and carbon tetrachloride (CCl4)-stimulated liver damaged mice in vivo and to determine whether or not this effect occurs by activating SIRT3. The results showed honokiol protects t-BHP-injured AML12 hepatocytes and CCl4-stimulated liver damage in mice by activating SIRT3. Honokiol reduces the acetylation level of superoxide dismutase 2 to enhance its anti-oxidative capacity, which decreases reactive oxygen species accumulation in AML12 cells. Honokiol increases the deacetylated peroxisome proliferator-activated receptor gamma coactivator 1-alpha level to promote mitochondrial biogenesis. Moreover, honokiol attenuates t-BHP induced mitochondrial fragmentation through Ku70-dynamin-related protein 1 axis. These results suggest that honokiol can ameliorate oxidative damage in hepatocytes by activating SIRT3, which might be a potential therapeutic agent for liver oxidative injury.