In recent years, Enterocytozoon hepatopenaei (EHP) has continuously caused outbreaks in global penaeid shrimp farming, resulting in stunted growth of penaeid shrimp and a substantial decline in production. In this study, artificial challenge was conducted by feeding the hepatopancreas tissue of EHP-positive penaeid shrimp. Subsequently, multiple techniques and methods, including electron microscopy, Oil Red O staining, biochemical index determination, real - time quantitative fluorescence PCR (RT-qPCR), and high-performance liquid chromatography, were employed to explore the mechanism of lipid metabolism imbalance in Litopenaeus vannamei induced by EHP infection at multiple levels, such as phenotype, gene, and metabolism. Electron microscopy revealed numerous lipid droplets of varying sizes within the interstitium of hepatopancreatic cells in EHP-infected shrimp. Oil Red O staining demonstrated pronounced lipid droplet accumulation in the hepatopancreatic tissue after two weeks of EHP infection. Biochemical analysis indicated that triglyceride (TG) levels were significantly elevated during the early infection stage (weeks 2), while total cholesterol (T-CHO) followed a trend similar to the control group throughout the infection period. Furthermore, lipoprotein lipase (LPL) activity in the hepatopancreas of infected shrimp was significantly lower than controls at weeks 3 and 4 post-infection, whereas hepatic lipase (HL) activity exhibited a compensatory increase. RT-qPCR analysis of lipid metabolism-related gene expression revealed significant upregulation of lipogenesis-associated genes (FAS, ACC) and downregulation of genes involved in lipolysis and fatty acid β-oxidation (CPT1, AMPKα, ACDVL, ACDM, ACBP, ACDS). HPLC analysis showed a 69.2% decrease in total fatty acid content, yet the proportions of long-chain polyunsaturated fatty acids (arachidonic ARA, EPA, DHA) were elevated. Collectively, these findings demonstrate that EHP disrupts the energy metabolic cycle in L. vannamei by interfering with lipid synthesis and fatty acid catabolism, suggesting potential diversion of host energy resources to support its own growth and development.