Abstract: This study investigated the time-dependent regulatory mechanisms of 8∶2 polyfluoroalkyl phosphate diester (8∶2diPAP) on lipid metabolism in zebrafish, with particular emphasis on the dynamic response patterns of the de novo lipogenesis pathway. A 28-day exposure experiment was conducted with a blank control group (CK) and three exposure groups treated with 8∶2diPAP, 8∶2 fluorotelomer carboxylic acid (8∶2FTCA), and perfluorooctanoic acid (PFOA), respectively, including 14 days of exposure followed by 14 days of depuration. Hepatic triglyceride (TG) and total cholesterol (TC) contents, as well as the levels of fatty acid synthase (FAS), stearoyl-CoA desaturase (SCD), and hormone-sensitive lipase (HSL), were determined. In addition, the expression of lipid metabolism-related genes, including elongation of very long-chain fatty acids protein 6 (ELOVL6), was analyzed. The results showed: compared with the blank control group, the hepatic TG and TC contents in zebrafish increased by 4.72-fold and 10.42-fold, respectively, and the perturbation intensity was greater than that induced by 8∶2FTCA and PFOA. Lipid synthesis-related molecules, including FAS, SCD, and ELOVL6, were upregulated, whereas HSL and carnitine palmitoyltransferase (CPT) were downregulated, suggesting a lipid metabolism shift from catabolism to anabolism. Pathway analysis indicated that 8∶2diPAP may promote de novo lipogenesis through activation of liver X receptor (LXR)-related signaling pathway. In conclusion, 8∶2diPAP exposure disrupted hepatic lipid metabolism in zebrafish, mainly reflected by enhanced de novo lipogenesis and suppressed lipid breakdown and fatty acid oxidation, ultimately leading to abnormal lipid accumulation. This process may be associated with activation of the LXR-related signaling pathway. By investigating the time-dependent regulatory mechanisms of 8∶2diPAP on lipid metabolism, this study provides a theoretical basis for elucidating its hepatic lipotoxic effects.