Novel DHA-enriched oils with high α-linolenic acid (ALA) content will be available in the near future as an alternative for dietary fish oil replacement in aquafeeds. As preliminary validation, we 1) assessed the ability of a diet containing a formulated oil blend (tuna oil + flaxseed oil, TOFX) with high DHA and ALA content to achieve fish oil-like omega-3 long-chain (≥C20) polyunsaturated fatty acids (n-3 LC-PUFA) tissue composition in Atlantic salmon smolts, and 2) applied liver proteomics as exploratory approach to understand the consequent nutritional changes. Comparisons were made on fish fed a fish oil-based diet (FO) and a commercial-like oil blend diet (fish oil + poultry oil, FOPO) over 89 days. Growth and feed efficiency ratio were lower on the TOFX diet. Fish tissue concentration of n-3 LC-PUFA and the n-3:n-6 ratio were significantly higher for TOFX than for FOPO, but not higher than for FO, while tissue retention efficiency of n-3 LC-PUFA was promoted by TOFX relative to FO. Proteomics analysis revealed an unexpected oxidative stress response as the main adaptive physiological mechanism in TOFX fish. While specific dietary fatty acid concentrations and balances and antioxidant supplementation may need further attention, the use of an oil with a high content of DHA and ALA can enhance tissue deposition of n-3 LC-PUFA in relation to a commercially used blend oil.