Perfluorooctanoic acid (PFOA), a typical perfluoroalkyl group compound, has received worldwide attention due to its significant environmental toxicity. Following regulatory bans on the production and emission of PFOA, concerns have been raised about the potential health risks and the safety of novel perfluoroalkyl analogues. HFPO-DA (trade name Gen-X) and HFPO-TA are two perfluoroalkyl analogues known to be bioaccumulative, whose level of toxicity and whether they are safe alternatives to PFOA remain unclear. In the following study, the physiological and metabolic effects of exposure to PFOA and its novel analogues were explored in zebrafish using 1/3 LC₅₀ (PFOA 100 μM, Gen-X 200 μM, HFPO-TA 30 μM). At the same LC₅₀ toxicological effect, exposure to PFOA and HFPO-TA resulted in abnormal phenotypes such as spinal curvature, pericardial edema and aberrant body length, while Gen-X was little changed. Metabolically, PFOA, HFPO-TA and Gen-X all significantly increased total cholesterol in exposed zebrafish with PFOA and HFPO-TA also increasing total triglyceride levels. Transcriptome analysis showed that the number of differentially expressed genes in PFOA, Gen-X, and HFPO-TA treated conditions compared to control groups were 527, 572, and 3, 933, respectively. KEGG and GO analysis of differentially expressed genes revealed pathways and functions related to lipid metabolism as well as significant activation of the peroxisome proliferators-activated receptor (PPARs) pathway. Furthermore, RT-qPCR analysis identified significant dysregulation in the downstream target genes of PPARα, which is responsible for lipid oxidative catabolism, and the SREBP pathway, which is responsible for lipid synthesis. In conclusion, both perfluoroalkyl analogues HFPO-TA and Gen-X exhibit significant physiological and metabolic toxicity to aquatic organisms and their environmental accumulation should be closely regulated.