Scutellaria baicalensis has been reported to improve the lipid metabolism of high-fat diet-induced liver dysfunction, but direct evidence is rare. This study aimed to explore the effects and mechanisms of S. baicalensis and its major constituent baicalin on hepatic lipotoxicity. KK-A mice and orotic acid (OA)-induced nonalcoholic fatty liver disease (NAFLD) rats were used to evaluate lipid metabolism regulatory effects. Sodium oleate-induced triglyceride-accumulated HepG2 cells were used for the mechanism study, pretreated with or without compound C or STO-609 or transfected with liver kinase B1 (LKB1) siRNA. In KK-A mice, S. baicalensis extract showed a decreased effect on serum and hepatic triglycerides, total cholesterols, and free fatty acid (FFA) levels after 8 weeks of treatment. In OA-induced NAFLD rats, 18 days of treatment with baicalin significantly inhibited hepatic lipid accumulation, attenuating hepatocyte hypertrophy, vacuolization and necrosis. S. baicalensis and baicalin treatment significantly suppressed the sterol regulatory element binding protein-1c (SREBP-1c) transcriptional program with downregulation of gene and protein expression of SREBP-1c (both precursor and mature fraction) and acetyl-CoA carboxylase, fatty acid synthase and stearoyl-CoA desaturase, and upregulation of AMP-activated protein kinase (AMPK), carnitine palmitoyl transferase 1 and nuclear respiratory factor 2 in the liver. Furthermore, activation of AMPK by baicalin was observed to be relative to the increase in phosphorylation of calmodulin-dependent protein kinase kinase. Taken together, S. baicalensis conferred preventive effects against FFA-induced lipotoxicity through the AMPK-mediated SREBP signaling pathway.