Lipids are the second largest nutrient source of fish, and play an important role in nutrient metabolism. As the essential fatty acid, the main component of lipid, long chain highly unsaturated fatty acids (LC-HUFA) play an important role in regulating metabolism and maintaining cell morphology. The synthesis of LC-HUFA involves many biological processes such as fatty acid transport, de novo synthesis, β-oxidation, desaturation, and carbon chain elongation. A stable LC-HUFA metabolic pattern has formed through long-term evolution. Previous studies have shown that exogenous intake can increase LC-HUFA accumulation and regulate metabolism in juvenile fish, however, there are few studies on this aspect in larvae. Early nutritional programming can affect the metabolism of the body, accompanied by epigenetic regulation. It is therefore crucial to explore how to activate LC-HUFA synthesis limitations by early nutrition programming in fish, which are further regulated by epigenetic mechanisms. This study explored the effects of docosahexaenoic acid (DHA) nutrition programming on the viability, fatty acid deposition, and epigenetic modification of fatty acid metabolism in larvae of Cynoglossus semilaevis to provide a theoretical basis for the development of high-quality C. semilaevis fish. Artemia salina hatchlings were fortified with a DHA fortifier. Larvae of C. semilaevis fed with fortified A. salina were used as the experimental group, while those fed with unfortified A. salina were used as the control group. The larvae were cultured for 15 days post-hatching (15 dph), and the hatching rate, survival rate, malformation rate, and body length were recorded. The whole body fatty acid profile and gene expression of fatty acid metabolism of larvae were analyzed. The DNA methylation status of fads2 gene was analyzed. Survival rate and body length of the experimental group was significantly increased, and the malformation rate was significantly decreased (P＜0.05) at 15 dph. The contents of linoleic acid (LA), arachidonic acid (ARA), linolenic acid (LNA), DHA, n-6PUFA, and n-3PUFA increased significantly (P＜0.05). The expression levels of pparα, acc1, and fas genes were significantly decreased, fads2 and fabp1 were significantly increased, and there was no significant difference in the expression level of elovlα (P＞0.05). There was a CpG island from –750 bp to –1,050 bp from the fads2 transcription start site, and the total length of the fragment was 301 bp. There were 8 candidate CpG sites, of which 5 were significantly demethylated (P＜0.05). The fads2 promoter region was highly demethylated (P＜0.05). This study revealed that nutrition programming of larvae of C. semilaevis with DHA can improve the survival ability of larvae, enhancing LC-HUFA synthesis by promoting fatty acid transport and desaturation in larvae to improve fatty acid metabolism. Early nutritional programming is involved in the demethylation of fads2 promoter, which promotes the transcription of fads2 gene, one of the root causes of the increase in LC-HUFA. This study will aid in the development of high LC-HUFA quality larvae of C. semilaevis and provide a basis for efficient breeding of this species.