Lipid accumulation, oxidative stress, and inflammation in hepatocytes are features of nonalcoholic fatty liver disease (NAFLD). Garcinia biflavonoid 1a (GB1a) is a natural product capable of hepatic protection. In this study, the effect of GB1a on anti-inflammatory, antioxidant, and regulation of the accumulation in HepG2 cells and mouse primary hepatocytes (MPHs) was investigated, and its regulatory mechanism was further explored. The result showed that GB1a reduced triglyceride (TG) content and lipid accumulation by regulating the expression of SREBP-1c and PPARα; GB1a reduced reactive oxygen species (ROS) and improved cellular oxidative stress to protect mitochondrial morphology by regulating genes Nrf2, HO-1, NQO1, and Keap1; and GB1a reduced the damage of hepatocytes by inhibiting the expression of inflammatory cytokines interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor-alpha (TNF-α), and nuclear factor kappa B (NF-κB) p65. The activities of GB1a were lost in liver SIRT6-specific knockout mouse primary hepatocytes (SIRT6-LKO MPHs). This indicated that activating SIRT6 was critical for GB1a to perform its activity, and GB1a acted as an agonist of SIRT6. It was speculated that GB1a may be a potential drug for NAFLD treatment.