The roles of long-chain polyunsaturated fatty acids (LC-PUFA) in osmoregulation and salinity adaptation in teleost fish were investigated in red tilapia (Oreochromis mossambicus♀ × O. niloticus♂). Groups of juvenile fish were reared initially in freshwater (FW, 0 ppt) or seawater (SW, 32 ppt) and fed diets containing either LC-PUFA-rich fish oil (FO) or rapeseed oil (VO) that lacks LC-PUFA, but is rich in C18 PUFA. After 8 weeks of feeding, the four groups of fish were subjected to salinity stress by directly changing to brackish water (BW, 16 ppt). Fish were sampled after 0 h, 3 h, 6 h, 1 d, 4 d, 8 d, and 16 d, and plasma osmoregulation-related parameters and tissue fatty acid compositions were analyzed. In red tilapia switched from SW to BW, plasma osmolality initially decreased significantly (0 - 1 d) and then increased (4 - 16 d) in fish fed the VO diet, while there were no significant changes in fish fed the FO diet. Plasma osmolality increased significantly in tilapia fed both diets when subjected to high salinity stress (FW to BW). Both the plasma Na concentration and gill activity of Na/K-ATPase (NKA) showed similar significant changes after salinity stress in fish fed the VO diet, but not in fish fed the FO diet. Plasma levels of growth hormone, cortisol, and prolactin were significantly increased after salinity stress in fish fed the VO diet, while prolactin level showed the opposite trend in fish fed the FO diet. Reflecting the fatty acid composition of the diets, LC-PUFA contents of gills and intestines were significantly higher in fish fed the FO diet than in fish fed the VO diet. Moreover, the liver expression levels of fads2 and elovl5 genes, which are involved in LC-PUFA biosynthesis, were significantly higher in red tilapia fed the VO diet than in fish fed the FO diet, and also in fish reared in seawater than in fish reared in freshwater. The time course of responses suggested that the adaptability of red tilapia to salinity stress was impacted by dietary LC-PUFA levels and that the FO diet provided the LC-PUFA required for fish to adapt rapidly to salinity changes and maintain osmotic balance, while fish fed the VO diet lacking LC-PUFA required the endogenous synthesis of LC-PUFA to adapt to acute salinity stress and maintain osmotic balance. Dietary LC-PUFA may influence plasma hormone levels and the activity of NKA in red tilapia through increasing cell membrane fluidity in order to respond to salinity stress and maintain osmotic equilibrium.