Abstract: Fish oil resources in aquafeeds are becoming increasingly scarce, and the extensive substitution of fish oil with vegetable oils often leads to abnormal lipid accumulation and metabolic dysfunction in fish livers. As a key intermediate linking carbohydrate and lipid metabolism, the role of lactate in this process warrants attention. Lactate dehydrogenase B (LDHB), which is responsible for oxidizing lactate to pyruvate, is a potential key node linking lactate metabolism and the reprogramming of hepatic fatty acid metabolism. In this study, we cloned the coding sequences (CDS) of the D. rerio ldhba and ldhbb genes and performed comprehensive bioinformatic analyses of their encoded proteins. We then characterized their tissue expression profiles and investigated the regulatory effects of different fatty acids and vegetable oil-substituted diets on ldhba and ldhbb expression through both in vitro and in vivo approaches.The results showed that the full-length CDS of D. rerio ldhba is 1 005 bp, encoding 334 amino acids with a molecular weight of approximately 36.24 ku and an isoelectric point (pI) of 6.39, while the ldhbb CDS is 1 068 bp, encoding 355 amino acids with a molecular weight of approximately 39.31 ku and a pI of 8.59. Both stable and hydrophobic proteins lack transmembrane domains, and their secondary structures are dominated by α-helices and random coils. Tissue distribution analysis revealed that both ldhba and ldhbb exhibited the highest expression levels in the liver. In vitro, treatment with 200-600 µmol/L of different fatty acids (linoleic acid, oleic acid, and palmitic acid) significantly downregulated the mRNA expression of both ldhba and ldhbb in D. rerio hepatocytes. In vivo, feeding a diet in which fish oil was completely replaced with palm oil and soybean oil for 30 days further confirmed a significant reduction in the hepatic expression levels of both genes. In conclusion, LDHB exhibits conserved molecular characteristics in D. rerio and its expression is robustly regulated by fatty acids. These findings suggest that LDHB may serve as a potential regulator in vegetable oil-induced hepatic lipid metabolism disorders in fish, thereby providing a theoretical foundation for formulating future nutritional intervention strategies.