Currently, the treatment of Metabolic dysfunction-associated fatty liver disease (MASLD) is limited, and this research aimed to investigate the therapeutic effect and molecular mechanism of Hydronidone (HDD) on MASLD. In our research, High fat and High cholesterol diet (HFHC) as well as Methionine-Choline Deficient diet (MCD) were used to establish the MASLD mouse model. Palmitic Acid (PA)-induced mouse primary hepatocytes (mpHCs) and AML12 cell line were used as cell model. The animal model was treated with three doses of HDD (25/50/100 mg/kg), and the cell model was treated with 0/50/100/200/400 μM HDD respectively. The therapeutic effect of HDD on MASLD models was observed by tissue staining, biochemical tests, qPCR, and Western Blot. Transcriptome sequencing was conducted among the normal control group (NCD group), model group (HFHC group) and medium-dose HDD treatment group (HFHC + 50 mg/kg HDD), through which CD36 was identified as a potential effector gene of HDD. Then, CD36 was overexpressed in the liver by adeno-associated virus on the basis of HFHC+HDD treatment model, and the effects of overexpression of CD36 on HDD treated MASLD mice were observed. The results showed mice in the HDD treatment group had significant improvements in liver steatosis, inflammation, and fibrosis compared with the model group (HFHC/MCD group). Lipid deposition in mpHCs and AML12 cells treated with PA was also significantly reduced after HDD intervention. The results of transcriptome sequencing showed that the expression of genes involved in lipid synthesis, inflammation, and fibrosis were significantly changed in the HDD treatment group. Overexpression of CD36 blocked the therapeutic effect of HDD on MASLD models. HDD could alleviate the disease progression by inhibiting the expression of CD36 in MASLD mice and cell models.