To explore the relationship between osmoregulation and long-chain polyunsaturated fatty acid(LC-PUFA) biosynthesis in rabbitfish (), an 8-week feeding trial was performed in juvenilerabbitfish with diets that contained fish oil (FO) or a blend of vegetable oils (VO; perilla and canola oils) at threesalinities: 10, 20, and 32. The growth performance, fatty acid composition of gill phospholipids, and activity andexpression levels of gill NaATPase (NKA) under different treatments were analyzed. The results showed that,at the same salinity, there was no effect of dietary lipid source on growth performance (0.05), whereas n-3LC-PUFA content in gill phospholipids in the FO dietary groups was significantly higher than those in VO treatments(0.05). In particular, although the contents of n-6 LC-PUFA in VO-fed fish were lower than in FO-fedfish, the opposite was observed for n-6 LC-PUFA contents. Furthermore, the activity and mRNA expression levelsof gill NKA in VO groups were significantly higher than those in FO groups (0.05). Under different salinities,the growth indexes at a salinity of 10 were significantly lower than those at salinities of 20 and 32 in both dietarygroups (0.05), whereas the LC-PUFA contents, and activity and mRNA expression levels of NKA in fish fedVO were significantly higher than that in fish fed VO (0.05). In addition, there were no significant differencesin either of the higher two salinities treatments (0.05). The results indicate that a salinity of 10 may not suitablefor rabbitfish culture. Furthermore, FO-derived diets can improve n-3 LC-PUFA content in gill phospholipids,which helps maintain membrane lipid fluidity and osmotic balance. Alternatively, VO-derived diets can regulateosmotic pressure by synthesizing n-6 LC-PUFA to up-regulate NKA expression and activity.