For a rapidly developing aquaculture industry where availability of raw materials for diet formulation are quickly changing it is fundamental to define what the dietary needs and benefits are. In farming of Atlantic salmon (Salmo salar), highly unsaturated fatty acids (HUFA) are known to be essential and a minimum dietary inclusion is needed. The aim of this study has been to map Atlantic salmon performance and midgut health towards dietary HUFAs under normal and challenging condition. Atlantic salmon post-smolt at 150 g were fed four experimental diets over a feeding period of 10 weeks up to a final weight of 377 g followed by three weeks of unpredictable chronic stress (UCS). The diets varied in their HUFA level, with either 28.7 g/kg DHA (HDHA), 25 g/kg EPA (HEPA), 21.3 g/kg ARA (HARA) or 8.5 g/kg EPA + DHA (LHUFA). After the feeding period all fish had more than doubled their weight and fish fed HARA showed a significant lower specific growth rate compared to all other diets. Diets had little effect on intestinal morphology, barrier and transport function. Intestinal polar lipid composition suggested higher lipid metabolism in fish feed LHUFA and transcriptome analysis confirm upregulated lipid metabolism and downregulated energy turn over. Comparing transcriptome profiles from HDHA to HEPA, more upregulated pathways are seen in fish fed HEPA, including metabolism and immunity. The fish feed HARA had downregulated turn-over and metabolism compared to fish fed HEPA. The UCS period resulted in zero growth and lower enterocyte height in all diet groups, and the intestinal barrier was reduced in the paracellular permeability to mannitol. Contradictory to previous studies, fish fed HDHA and HEPA responded to UCS with enhanced intestinal resistance. UCS also resulted in impaired intestinal transport by elevated ion flux and epithelial electrochemical gradient in fish fed LHUFA. Transcriptome pathway analysis indicated more cell turn over and lipid metabolism in LHUFA and HDHA. In conclusion, a low HUFA diet with 8.5 g/kg DHA + EPA can sustain growth and intestinal health under controlled condition, but the health is impaired after three weeks of chronic stress. Decreasing the n6/n3 ratio by increasing levels of EPA and DHA strengthens intestinal barrier in response to chronic stress. Increasing the ARA to HUFA ratio may have a negative effect on growth, but not on intestinal health.