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Long chain acyl-coA synthase (<i>acsl</i>) family genes activate the conversion of long chain fatty acids into acyl-coA to regulate fatty acid metabolism. However, the evolutionary characteristics, tissue expression and nutritional regulation of the <i>acsl</i> gene family are poorly understood in fish. The present study investigated the molecular characterization, tissue expression and nutritional regulation of the <i>acsl</i> gene family in golden pompano (<i>Trachinotus ovatus</i>). The results showed that the coding regions of <i>acsl1</i>, <i>acsl3</i>, <i>acsl4</i>, <i>acsl5</i> and <i>acsl6</i> cDNA were 2091 bp, 2142 bp, 2136 bp, 1977 bp and 2007 bp, encoding 697, 714, 712, 659 and 669 amino acids, respectively. Five <i>acsl</i> isoforms divided into two branches, namely, <i>acsl1</i>, <i>acsl5</i> and <i>acsl6</i>, as well as <i>acsl3</i> and <i>acsl4</i>. The tissue expression distribution of <i>acsl</i> genes showed that <i>acsl1</i> and <i>acsl3</i> are widely expressed in the detected tissues, while <i>acsl4</i>, <i>acsl5</i> and <i>acsl6</i> are mainly expressed in the brain. Compared to the fish fed with lard oil diets, the fish fed with soybean oil exhibited high muscular C₁₈ PUFA contents and <i>acsl1</i> and <i>acsl3</i> mRNA levels, as well as low muscular SFA contents and <i>acsl4</i> mRNA levels. High muscular n-3 LC-PUFA contents, and <i>acsl3</i>, <i>acsl4</i> and <i>acsl6</i> mRNA levels were observed in the fish fed with fish oil diets compared with those of fish fed with lard oil or soybean oil diets. High n-3 LC-PUFA levels and DHA contents, as well as the <i>acsl3</i>, <i>acsl4</i> and <i>acsl6</i> mRNA levels were exhibited in the muscle of fish fed diets with high dietary n-3 LC-PUFA levels. Additionally, the muscular <i>acsl3</i>, <i>acsl4</i> and <i>acsl6</i> mRNA expression levels, n-3 LC-PUFA and DHA levels were significantly up-regulated by the increase of dietary DHA proportions. Collectively, the positive relationship among dietary fatty acids, muscular fatty acids and <i>acsl</i> mRNA, indicated that <i>T. ovatus</i> Acsl1 and Acsl3 are beneficial for the C₁₈ PUFA enrichment, and Acsl3, Acsl4 and Acsl6 are for n-3 LC-PUFA and DHA enrichment. The acquisition of fish Acsl potential function in the present study will play the foundation for ameliorating the fatty acids nutrition in farmed fish products.