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Terrestrial protein and lipid sources are commonly used as substitutes for marine fishery-derived raw ingredients in fish diets. However, their use is related with several side-effects on marine fish performance, health, or disease resistance. Physical barriers of the skin, gills, and gut constitute the primary defense mechanism of fish. Skin mucus mucosal mucins, water, proteins, ions, and lipids determine the physical, chemical, and protective characteristics of skin mucus. Very little is known about the influence of diet composition on fish skin mucus fatty acid profile. Gilthead sea bream skin mucus contained 10% of total lipids (TL), which consisted of 50⁻60% neutral (NL) and 40⁻50% polar lipids (PL) fractions. Σn−3 long chain polyunsaturated fatty acids (LC-PUFA) deposition was preferential in the NL fraction, whereas Σn−6LC-PUFA accumulation was similar in both lipid classes. Docosahexaenoic acid (DHA; 22:6n−3) was the main LC-PUFA stored in skin mucus (14% TL) in relation to eicosapentaenoic acid (EPA; 20:5n−3) (2⁻3% TL) and arachidonic acid (ARA; 20:4n−6) (2% TL). This study denotes the importance of DHA as component of skin mucus lipids compared to other essential fatty acids, such as EPA and ARA, as well as importance of maintaining an adequate Σn−3/ Σn−6 ratio, regardless of dietary intake.