Objective To investigate the key signal molecules in the regulation of biological clock and lipid metabolism of non-alcoholic fatty liver disease（NAFLD）and its effect on lipid metabolism, to provide insights for the prevention and treatment of NAFLD. Methods The animal models with NAFLD were established and classified, including circadian rhythm disorder + high-fat diet(HFC) group, circadian rhythm disorder + normal diet(NC) group, high-fat diet (HF) group and normal diet (N) group. Hematoxylin and eosin (HE) and oil red O staining were used to detect the fat deposition in the model liver tissues; enzyme-linked immunosorbent assay(ELISA) was used to detect the serum lipids in the mice; immunoblotting was performed to detect the protein expression of brain and muscle arnt-like 1(BMAL1) gene，and the correlation between BMAL1 gene and the liver pathological features was estimate. For the HFC and HF groups, mRNA bioinformatics analysis was performed to identify key circadian clock genes in NAFLD. Results Circadian rhythm disturbance increased body weight and induced obesity in mice. At week 15, the HFC group showed significantly higher weight than the HF group (t=23.18, P<0.000 1), and the NC group exceeded the N group (t=5.24, P<0.000 1). It also promoted hepatic lipid deposition: lipid content progressively increased in the HFC group (F=10.13, P<0.05) and NC group (F=8.89, P<0.05) over time. Moreover, it exacerbated dyslipidemia: TC and LDL-C levels in the HFC group were significantly higher than the HF group at ZT0, ZT8 and ZT16 (F=23.3, P<0.0001; F=68.1, P<0.000 1); similarly, the NC group had elevated TC and LDL-C versus the N group (F=3.9, P<0.000 1; F=5.8, P<0.000 1). BMAL1 expression exhibited rhythmic fluctuations, with higher protein levels at ZT16 than ZT8 in HFC and NC groups, showing a significant positive correlation with fatty liver severity (r=0.995, P=0.022). Conclusions A high-fat diet causes abnormal lipid metabolism in mice; the disturbance of circadian rhythm exacerbates the abnormal lipid metabolism in mice, increases lipid deposition in the liver, and promotes the progression of fatty liver. The biological clock gene BMAL1 is closely related to the metabolism of non-alcoholic fatty liver disease. High expression of BMAL1 may induce fat accumulating in the liver.