The control of feed intake in fish in aquaculture requires the development of new techniques to improve diet composition, feed conversion efficiency and growth. The aim must be sustainability and an effective use of resources. The effect of replacing traditional aqua-feed ingredients (fishmeal and fish oil) by a 100% plant-based diet is known to drastically decrease fish performance (survival and growth). One disadvantage of the use of plant ingredients relies on the modification of the nutrient composition of the diet with totally absence of essential ω-3 long chain polyunsaturated fatty acids (ω-3 LC-PUFAs), mainly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). In fish ω-3 LC-PUFAs are known to be essential in their life cycle, promoting optimal growth and survival, health, reproduction and offspring development. In farmed fish, except for a study about the effect on the origin of the fat source on feed selection in fish, the specific role of lipids and particularly ω-3 LCPUFA on feeding behavior (preference, food intake and uneaten food) has not yet been investigated. This information is crucial to understand if the decrease of survival rate and growth performance of fish fed with plant-based diets could be explained by the modification of feeding behavior due to the absence of ω-3 LC-PUFAs. Materials and Methods Using self-feeder, the present study examined the feed preference of rainbow trout Oncorhynchus mykiss for three diets containing distinct levels of omega-3 long chain polyunsaturated fatty acids (ω-3 LCPUFA): eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (0% for low, 5% for medium and 20% for high, total fatty acid content). Feed preference values for each group (low v. medium ω-3 diets, medium v. high ω-3 diets and low v. high ω-3 diets) were observed using two self-feeders positioned at opposite sides of the tank. The hypothesis was that the decrease of fish growth and survival rate of fish fed with 100% plant-based diet could be explained by the absence of ω-3 LCPUFA relating to decrease of food intake. This could explain the tasting role of ω-3 LCPUFA in the feeding behavior of rainbow trout (which reflects the motivation to consume feed). Results The results showed that rainbow trout could discriminate between the diets containing different level of ω-3 LCPUFA even if unable to differentiate between level of 5% (no preference observed in low v. medium ω-3 diets). Overall they had a preference for diet high in ω-3 LCPUFA: 59.5% preference for high ω-3 diet in high v. low ω-3 diets, and 75.6% preference for high ω-3 diet in medium v. high ω-3 diets respectively. In parallel, the composition (selected enzymatic and non-enzymatic oxidation metabolites) and the impact of dietary ω-3 LC-PUFAs on central molecular mechanisms regulating feeding behavior was determined. Total selected enzymatic oxidation metabolites of DHA and EPA increased in high ω-3 LC-PUFAs diet where as total selected non-enzymatic oxidation metabolites of DHA (not detected for EPA) increased in medium and high ω-3 LC-PUFAs diets. Overall transcript genes related to pro-inflammatory cytokines, inflammation, antioxidant status, cortisol pathway, serotoninergic pathways and dopaminergic pathways were down-regulated in the juveniles trout fed with the high ω-3 LC-PUFAs diet. Conclusion In conclusion, our data revealed that a diet rich in ω-3 LC-PUFAs drove the feeding behaviour of juvenile rainbow trout with affected a relatively high proportion of the brain function and composition in rainbow trout through mechanisms comparable to those characterized previously in mammals.