Excessive deposition of fish fat is a very common phenomenon in artificially farmed fish. The widespread use of high-fat diets is an important cause for this consequence. Long-term and excessive use of high-fat diets can cause fat deposition in fish organs or tissues. Liver cells are the main place for fat synthesis in fish, and 90% of the fat is synthesized in liver cells. Excessive fat deposits in the liver can have a negative impact on the health of the fish. Chinese herbal medicine is the current research focus of anti-immune stress agents. Berberine is an alkaloid isolated from the traditional Chinese medicine Rhizoma Coptidis. It has the effects of protecting the liver, antioxidant, anti-inflammatory, hypoglycemia and lipid-lowering. Preliminary research in our laboratory found that berberine as a functional feed additive can significantly inhibit the process of oxidative stress, reduce cell apoptosis and enhance the immunity of fish fed with high-fat diets. In order to explore the effect of berberine on the lipid metabonomics of Danio rerio liver cells induced by high fat, D. rerio cells were used as experimental materials, and sodium palmitate (0.25 mmol/L, 24 h) was used to induce liver cell lipid deposition models. After treatment with berberine (25 μmol/L, 6 h), the cells were collected. Using high performance liquid chromatography-mass spectrometry technology for lipid metabolomics analysis, the lipid metabolites of different treatments of D. rerio liver cells were screened and identified, and the gene expression of lipid metabolism-related factors was detected. The results showed that compared with the control group, a total of 1 761 differential ions were screened in the high-fat group, among which 147 differential metabolites were identified, and 123 were increased and 24 were decreased; the high-fat + berberine group screened a total of 2 728 differential ions, among which 346 different metabolites were identified, 139 increased and 207 decreased. Compared with the high-fat group, the high-fat + berberine group screened a total of 1 605 differential ions, of which 259 differential metabolites were identified, and 45 increased and 204 decreased. Further studies on the metabolic pathways of unsaturated fatty acids found that the content of differential metabolites dihomo-γ-linolenic acid (DGLA) and dihomo-α-linolenic acid (ETA) increased significantly in the high-fat group, and the addition of berberine would make their content decrease significantly; compared with the control group, there was no significant difference in the content of DGLA and ETA in the high-fat + berberine group. By detecting the expression of DGLA and ETA decomposition-related enzyme genes (atgl, hsl), it was found that compared with the control group, both of the high-fat group were significantly reduced, and both were significantly increased after the addition of berberinei; detection of the expression levels of synthesis-related enzyme genes (acc, elovl6, elovl7a, scdb, fas and fads2), showed that compared with the control group, the high-fat group intracellular acc, elovl6 and scdb expression were significantly increased, fads2 expression decreased significantly, and fads2 expression in cells increased significantly after berberine was added, and fas, elovl6 and scdb expressions were significantly decreased. To sum up, the induction of high fat inhibits the expression of lipid-related genes, promotes the expression of lipid-synthesis-related genes, and causes excessive deposition of fat in cells. Adding a proper amount of berberine can promote the expression of lipid-related genes and inhibit the expression of lipid synthesis-related genes, thereby down-regulating the content of DGLA and ETA, effectively alleviated the damage induced by sodium palmitate to liver cells.