It has been reported that the histone/protein deacetylase SIRT1-AMP-activated protein kinase (SIRT1-AMPK) signaling pathway may play a role in the effects of dihydromyricetin (DHM) on improving triglyceride (TG) accumulation and insulin resistance in liver cells. Therefore, we aimed to further observe the effect of DHM on liver fat deposition in high-fat diet (HFD)-induced obese mice and explore its possible mechanism. C57BL/6J mice were fed with a normal diet (ND) and HFD and were treated with or without low-dose (125 mg/kg) or high-dose (250 mg/kg) DHM for 16 weeks, respectively. During the experiment, body weight was checked every 2 weeks. After 16 weeks, the orbital vein was bled, the animals were sacrificed, and the subscapular, epididymal, and inguinal fat were collected and weighed with an electronic scale. An automatic biochemical analyzer was used to determine the levels of serum triglyceride (TG), serum total cholesterol (TC), serum high-density lipoprotein (HDL), and serum low-density lipoprotein (LDL). The livers were stained with hematoxylin-eosin staining (H&E) and Oil Red O to detect liver fat deposition. A colorimetric method was used to detect liver MDA and SOD contents. Quantitative real-time PCR (qRT-PCR) was used to detect the gene expressions of related indicators, such as interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor-α (TNF-α), acetyl-CoA carboxyl acetyl-CoA carboxylase (ACC), sterol regulatory element-binding protein-1c (SREBP-1), fatty acid synthetase (FAS), peroxisome proliferator activation receptor alpha (peroxisome proliferator-activated receptor-alpha, PPARα), palmitoyltransferase 1 (carnitine palmitoyltransferase 1, CPT1), SIRT1, and AMPK. Western blotting analysis was used to detect the protein expression levels of SIRT1, AMPK, SIRT1-AMPK, ACC, SREBP-1, FAS, PPARα, and CPT1. Results showed that compared with the ND group, the weight and body fat of the mice in the HFD group were increased significantly. The levels of TG, TC, and LDL were increased, the level of HDL was decreased, the volume of hepatocytes was increased, the number of lipid droplets, fat deposition, MDA, IL-6, IL-8, TNF-α, SREBP-1c, FAS, ACC1, SIRT1, and AMPK protein levels were significantly increased, and the SOD activity, PPARα, CPT1, SIRT1 mRNA, AMPK mRNA, PPARα, CPT1 levels were significantly decreased. DHM could significantly reverse the changes of the above indexes in HFD mice, while DHM had no significant effect on the above indexes in ND mice. Collectively, our findings revealed that DHM improved liver fat deposition in HFD-induced obese mice, and the mechanism might be related to inhibition of oxidative stress, inflammation, lipid synthesis, and promotion of lipid decomposition.