Evidences of oxidative stress and mitochondrial dysfunction have been recognized in most of clinical and experimental liver diseases. SIRT3, a member of NAD+-dependent deacetylases, is mainly localized in mitochondria. So far, the role of SIRT3 in protecting hepatocytes against oxidative stress remains elusive. Herein, we found SIRT3 protein expression is decreased in tert-butyl hydroperoxide (t-BHP)-treated AML12 cells in vitro and primary hepatocytes from CCl4-injured mice in vivo. To further verify the role of SIRT3 in protecting hepatocytes from t-BHP-induced injury, SIRT3 overexpressed AML12 cell line and primary hepatocytes were generated. SIRT3 overexpressed hepatocytes showed improved cell viability upon t-BHP challenge, with less intracellular reactive oxygen species (ROS) accumulation. SIRT3 overexpression reduced superoxide dismutase 2 acetylation level and stimulated nuclear factor erythroid 2-related factor 2 nuclear translocation to enhance anti-oxidative capacity. Moreover, SIRT3 deacetylated peroxisome proliferator-activated receptor γ coactivator 1α to promote mitochondrial biogenesis, and 8-oxoguanine DNA glycosylase 1 to orchestrate DNA repair, resulting in improved mitochondrial function. Through deacetylating Ku70, SIRT3 also abated mitochondrial translocation of dynamin-related protein 1, to attenuate mitochondrial fragmentation in t-BHP-injured hepatocytes. These results suggested that SIRT3 protected hepatocytes against oxidative stress by enhancing ROS scavenging and maintaining mitochondrial integrity.