Disruptions to circadian rhythm have been associated with an increased risk of nonalcoholic fatty liver disease (NAFLD). DHA has been found to affect both circadian rhythm and lipid metabolism. In this study, the relationship between DHA substitution and improvements in lipid metabolism and circadian clock regulation was studied. Male C57BL/6 mice were fed a control, a high fat or a DHA substituted diet for 12 weeks. Biochemical analysis and H&E staining showed that the high-fat diet (HFD) could induce NAFLD, and DHA substitution (AOH) could attenuate NAFLD. The qPCR results showed that the expressions of core clock genes Clock and Bmal1 were significantly higher at zeitgeber (ZT) 0 (7:00 am) than those at ZT12 (7:00 pm) in the control group, while this difference in day and night disappeared in the HFD group, but was observed in the AOH group. Western blotting results indicated that the expressions of rhythm output molecules (RORα and REV-ERBα) and their downstream protein INSIG2 all showed the corresponding circadian changes. SREBP-regulated proteins were significantly increased in the HFD group at both ZT0 and ZT12, but decreased in the AOH group accompanied by the corresponding changes in the protein expressions of HMGCR, LXR, CYP7A1 and CYP27A1. Altogether, HFD can decrease or disrupt circadian rhythm fluctuation by up-regulating the expression of core circadian rhythm genes Clock and Bmal1 at ZT12, and induce metabolic abnormalities through the INSIG2-SREBP pathway regulated by RORα and REV-ERBα. DHA substitution seems to restore circadian rhythm similar to the normal circadian rhythm of "night-high, day-low" through the metabolic pathway regulated by rhythmic nuclear receptors, improving the lipid metabolism rhythm and reducing liver fat.