3-hydroxyacyl-CoA dehydratases 1 (Hacd1) is a critical enzyme in long-chain polyunsaturated fatty acids (LC-PUFA) biosynthesis. The difference in expression of hacd1 might account for the stronger capacity of LC-PUFA biosynthesis in freshwater fish than in marine fish, but little is known about fish hacd1. Therefore, this study compared the responses of large yellow croaker and rainbow trout hacd1 to different oil sources or fatty acids, and also examined transcriptional regulation of this gene. In this study, hacd1 was highly expressed in the liver of large yellow croaker and rainbow trout, which is the main organ for LC-PUFA biosynthesis. Therefore, we cloned the hacd1 coding sequence, with a phylogenetic analysis showing that this gene is evolutionarily conserved. Its localization to the endoplasmic reticulum (ER), likely also indicates a conserved structure and function. The expression of hacd1 in the liver was significantly decreased after the substitution of soybean oil (SO) for fish oil but was not significantly affected after palm oil (PO) substitution. Linoleic acid (LA) incubation significantly promoted hacd1 expression in primary hepatocytes of large yellow croaker and eicosapentaenoic acid (EPA) incubation significantly promoted hacd1 expression in primary hepatocytes of rainbow trout. Transcription factors STAT4, C/EBPα, C/EBPβ, HNF1, HSF3 and FOXP3 were identified in both large yellow croaker and rainbow trout. HNF1 had a stronger activation effect in rainbow trout than in large yellow croaker. FOXP3 inhibited hacd1 promoter activity in large yellow croaker but had no effect in rainbow trout. Therefore, the differences between HNF1 and FOXP3 affected the expression of hacd1 in the liver thus being responsible for the high capacity of LC-PUFA biosynthesis in rainbow trout.