Long‐chain polyunsaturated fatty acids (LC‐PUFAs) play crucial roles in growth, development and reproduction of Litopenaeus vannamei, one of the most important shrimp species cultivated in China in recent years. To investigate the effect of dietary n‐3 PUFA composition on growth and the expression of LvFAD6 and LvELOV5 genes and fatty acid composition of this species, ten diets, which contain different levels of α‐linolenic acid (C18:3 n‐3, ALA), eicosapentaenoic acid (C20:5 n‐3, EPA) and docosahexaenoic acid (C22:6 n‐3, DHA), respectively, were prepared for the culture of this species lasting 42 days. The results showed that the identified cDNA sequence of fatty acyl desaturases 6 from L. vannamei (LvFAD6) was 739 bp, encoding a protein of 210 amino acids. The LvFAD6 peptide sequence possessed two histidine‐rich motifs (QHHA and QIEHH), shared 96.6% identity with Apostichopus japonicus FAD6 and strongly clustered with FAD6 from A. japonicus and Octopus bimaculoides. The expression of LvFAD6 and LvELOV5 in muscle decreased with increase in dietary EPA, but increased with increase in dietary DHA. Dietary ALA has the potential to further increase the production of these health‐beneficial LC‐PUFAs in L. vannamei fed a diet with low levels of EPA and DHA, implying L. vannamei had the capacity to convert dietary ALA into EPA and DHA. These results suggest that the supplementation of ALA, EPA and DHA in diets for better growth of L. vannamei in ranges of 2.5% to 3.89%, 1.7% to 3.19% and 2.7% to 5.84% of total fatty acids respectively.