Abstract: In order to explore the role of fatty acid elongase 4b (elovl4b) gene in the synthesis of long-chain polyunsaturated fatty acids (LC-PUFAs) in freshwater fish, we utilized the CRISPR/Cas9 system to construct a gene mutation model with a 2-base deletion in exon 2 of the elovl4b^–/– gene. The fatty acid composition of liver in elovl4b zebrafish was analyzed, revealing a significant increase in the total fatty acids and C22PUFA compared to wild-type (WT) zebrafish. Additionally, the proportions of various LC-PUFAs components showed significant changes. Further transcriptome sequencing of the zebrafish liver was conducted, resulting in the creation of gene libraries and the identification of differentially expressed genes. Enrichment analysis and functional annotation were performed using database resources, and significant differentially expressed genes in the KEGG pathway were verified by qPCR. The results showed substantial changes in gene expression profiles, and 809 genes showing significant differences in expression. Pathways such as the pentose phosphate pathway were notably enriched, while pathways involved in fatty acid synthesis were significantly up-regulated. Conversely, arachidonic acid metabolism and primary bile acid synthesis pathways were significantly down-regulated. Validation of twelve randomly selected genes using Real-time Quantitative PCR (qPCR) demonstrated consistency with the transcriptome sequencing results. These findings suggest that the deletion of elovl4b induces adaptive changes in critical biological processes, including arachidonic acid metabolism, fatty acid metabolism, and fat production and metabolism. This study provides insights into the role of elovl4b in LC-PUFAs synthesis and offers a theoretical basis for treating related cardiovascular diseases, chronic obesity, and other conditions.