Abstract: Abstract   This study is aimed to explore the time-dependent regulatory mechanism of 8:2 polyfluoroalkyl phosphate diester (8:2diPAP) treatment on lipid metabolism in zebrafish, with a focus on analyzing the response patterns of the de novo lipid synthesis pathway. By established a control group (CK) and an 8:2diPAP exposure group, exposing the zebrafish to 8:2diPAP for a period of 0-14 days followed by a 15-28 day filtration stage, and sampling at days 7, 14, 21, and 28 to systematically evaluate the time-dependent changes in lipid metabolism. By measuring liver triglyceride (TG) and total cholesterol (TC) levels, combined with the expression levels of key functional molecules such as fatty acid synthase (FAS), stearoyl-CoA desaturase (SCD), and hormone-sensitive lipase (HSL), as well as lipid metabolism-related genes including FAS, elongase 6 (ELOVL6), and others, we elucidated the regulatory effects of 8:2 diPAP on the lipid metabolism network. (ELOVL6), the regulatory effects of 8:2diPAP on the lipid metabolism network were analyzed. Exposure to 8:2diPAP disrupted lipid metabolism dynamics: phenotypically, TG and TC content showed a continuous accumulation trend with exposure time; Functional molecular results indicate that levels of lipid synthesis-related molecules (FAS, SCD) increased, while levels of lipolysis (HSL) and fatty acid β-oxidation-related molecules Carnitine palmitoyl transferase (CPT) decreased. Genetic test results indicate that expression of key genes for de novo lipid synthesis (FAS, ELOVL6) was upregulated, while expression of genes related to lipid catabolism (HSL, CPT) was downregulated. Further analysis combined with signaling pathway analysis revealed that this regulatory process may involve pathways such as liver X receptors (LXR) and sterol regulatory element-binding protein (SREBP), indicating that 8:2diPAP drives abnormal lipid accumulation by promoting de novo lipid synthesis and inhibiting lipid decomposition and oxidation, thereby inducing hepatotoxic effects. The results of this study indicate that exposure to 8:2diPAP can significantly disrupt the lipid metabolism homeostasis in the liver of zebrafish. With prolonged exposure time, TG and TC content continue to increase, and the expression of lipid synthesis-related molecules and genes (such as FAS, SCD, ELOVL6) significantly enhances, while lipolysis and fatty acid β-oxidation-related molecules and genes (such as HSL, CPT) are inhibited. Based on comprehensive phenotypic, biochemical, and transcriptional changes, it is evident that 8:2 diPAP promotes the shift from catabolism to anabolism of lipids by activating de novo lipid synthesis and inhibiting lipid decomposition and oxidation processes, ultimately leading to abnormal lipid accumulation. This provides new insights into the interference of perfluoroalkyl substances on lipid metabolism.