Hepatocyte nuclear factor 4 (HNF4) is a pivotal transcription factor that regulates lipid metabolism and the biosynthesis of long-chain polyunsaturated fatty acids (LC-PUFA) and plays a critical role in maintaining hepatocyte function. LC-PUFAs, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are essential for various physiological processes. However, most aquatic species exhibit limited biosynthetic capacity. Eriocheir sinensis, an economically significant crab species, serves as an important model for investigating lipid metabolism mechanisms in aquaculture. Despite its importance, the functional role of Hnf4 in crustaceans remains poorly understood. This study aims to elucidate the molecular characteristics and regulatory mechanisms of Hnf4 in E. sinensis. The full-length Hnf4 gene was cloned using rapid amplification of cDNA ends (RACE) technology, and its mRNA expression profile across multiple tissues was analyzed via quantitative polymerase chain reaction (qPCR). The full-length sequence of the Hnf4 gene in E. sinensis is 3554 bp, including a 5 ʹUTR of 569 bp, a 3ʹ UTR of 1179 bp, an ORF of 1806 bp, and Hnf4 cDNA encoding 601 amino acids. The overall average hydrophilicity and hydrophobicity of HNF4 protein is −0.177, indicating that the HNF4 protein in E. sinensis is a hydrophilic protein. Homology analysis revealed that the HNF4 of E. sinensis has high sequence and amino acid homology with crustaceans such as Procambarus clarkii, Scylla paramamosain and Portunus trituberculatus, and is clustered together with the HNF4 of Portunus trituberculatus and Procambarus clarkii in the phylogenetic tree. The qPCR results showed that Hnf4 was expressed in multiple tissues of E. sinensis, with the highest expression in the hepatopancreas. E. sinensis were treated with ds Hnf4, inhibitor BI-6015, and agonist Benfluorescex. After 24 h of treatment, hepatopancreatic tissue samples were collected to detect changes in the expression of lipid metabolism-related genes. The results showed that after injection of the inhibitor BI-6015 and interference of Hnf4 gene expression with dsRNA, the expression of Fas, Scd, Fad6, Fad9, and Elovl4 in the hepatopancreas of E. sinensis was significantly reduced. Meanwhile, the expression of Hsl was significantly upregulated (P＜0.05), whereas injection of the agonist Benfluorescex yielded the opposite result. This indicates that the Hnf4 gene plays a role in regulating lipid metabolism and LC-PUFA biosynthesis in E. sinensis. This study represents the first successful cloning of the Hnf4 gene in E. sinensis and demonstrates its regulatory role in LC-PUFA biosynthesis by modulating key lipid synthesis genes (Fas and Scd) and enzyme genes (Fad6, Fad9, Elovl4). The hepatopancreas has been identified as the primary site of Hnf4 activity, where its suppression leads to reduced fatty acid synthesis and enhanced oxidative metabolism. Notably, the relatively low expression of Hnf4 in the eyestalk of E. sinensis suggests potential species-specific regulatory mechanisms distinct from those observed in mammals. These findings provide valuable insights into the biological specificity of HNF4 and its physiological regulation of lipid metabolism in E. sinensis. Furthermore, they offer a theoretical foundation for elucidating the lipid metabolism network in crustaceans and for reducing the reliance of aquaculture on fish oil. Future research should focus on exploring the distribution of HNF4 subtypes and their interactions with other transcription factors.