Metabolic responses to sub-optimal temperature deplete lipid depots, remodel membrane lipid and alter the fatty acid profile in the whole body and tissues of ectothermic vertebrates including fish. The magnitude of these changes may depend on dietary history including oil sources with different fatty acid compositions. Barramundi, Lates calcarifer (Perciformes, Latidae), a tropical ectothermic fish, was fed on diets either rich in dietary long-chain (≥C(20)) polyunsaturated fatty acids (LC-PUFA) from fish oil, rich in stearidonic and γ-linolenic acid (SDA and GLA, respectively) from Echium plantagineum, or rapeseed oil deficient in LC-PUFA. Following 5 weeks at the optimum temperature of 30 °C when growth rates were comparable amongst dietary treatments, water temperature was dropped to 20 °C for 1 week for half of the animals and maintained at 30 °C for the other half. Decreased temperature increased the liver and skeletal muscle content of LC-PUFA in fish fed on echium oil compared with rapeseed oil, while dietary LC-PUFA depots in fish oil fed-fish depleted rapidly in the week of sub-optimal temperature. The lipid unsaturation index of cellular membrane in the liver and muscle increased under low temperature at the same rate regardless of dietary oil. Therefore, rapid exposure of an ectothermic vertebrate to a lower and sub-optimal temperature caused significant modulation in fatty acid composition. We propose that the tolerance of barramundi, a representative of tropical farmed fish, to sub-optimal temperature will be enhanced when fatty acid substrates closer to the LC-PUFA are available in their diet.