The synthesis of highly unsaturated fatty acids (HUFAs) from C18 fatty acids in the Pacific white shrimp, Litopenaeus vannamei, has long been debated in terms of the limited capacity or even the existence of fatty acid desaturase and elongase. In the present study, a Δ6 fatty acyl desaturase (Fad)-like gene was identified and characterized with a full length of 1,491 bp, including a 39-bp 5′-UTR (untranslated region), a 108-bp 3′-UTR, and a 1,344 bp open reading frame encoding a 448 amino acid protein. The protein contained a cytochrome b5-like heme/steroid binding domain and a Fad domain. The Δ6 Fad-like protein of L. vannamei shared 70%–77% sequence homology with those of Macrobrachium nipponense, Scylla paramamosain, Portunus trituberculatus and Eriocheir sinensis. When salinity was reduced from 30 to 3, Δ6 Fad-like gene expression significantly increased in the gills, muscles, eyestalk, and hepatopancreas (P < 0.05). The highest expression was observed in the hepatopancreas under salinity 3 in the linseed oil group, followed by the fish oil group. The results suggest that L. vannamei has a gene encoding a Δ6 Fad-like protein whose expression increases with decreased salinity and depends on the level of C18 fatty acids, such as α-linolenic acid and linoleic acid, in the diet. The present study improves the current understanding of Δ6 Fad-like genes in euryhaline shrimp species, particularly their mRNA expression under different conditions.