Aquaculture’s rapid growth and a diminishing supply of fish oil has led to a significant amount of research over the last decade conducted to replace fish oil with alternative lipid sources, primarily vegetable oils, which are more arachidonic acid (ARA) deficient than fish oil. ARA has a variety of physiological functions, one of the most important of which is that it serves as a primary precursor to eicosanoids that play crucial roles throughout the life cycle in several physiological processes including growth and development, immune function, stress and inflammatory responses, and reproduction. The objective of the study was to evaluate the effects of dietary ARA on growth performance, fatty acid composition of whole-body and ovary, nonspecific immunity, and intestinal inflammatory gene expression in rainbow trout Oncorhynchus mykiss. Four isonitrogenous (51%, crude protein), isolipidic (15%, crude lipid), and isocaloric (23 MJ/kg) diets were produced: ARA 0 (linseed oil, i.e., the control diet), and three other diets with graded levels of ARA-rich oil (ARA-L: 0.59%, ARA-M: 1.14%, and ARA-H: 2.29% of diets). The feeding trial was conducted in three phases. Phases 1 and 2 were performed to evaluate only growth and feed utilization. Phase 3 was performed to evaluate growth and feed utilization as well as fatty acid composition of whole-body and ovary, inflammatory responses, and non-specific immune responses in response to acute stress. In Phase 1, rainbow trout fry (0.17 ± 0.01) were fed to apparent satiation six times a day for 8 weeks to assess early growth performance and feed utilization. For Phase 2, 50 fish/tank were randomly selected from Phase 1 and continued to evaluate growth performance and feed utilization for another 16 weeks. In Phase 3, rainbow trout (307 ± 3.64) were fed to apparent satiation three times a day for 12 weeks to assess growth performance and feed utilization, then fish were exposed to an acute stress (lowering the water level to 5 cm, just above the eyes, for 30 mins). The growth performance and feed utilization of fish during Phase 1 (fry), Phase 2 (juvenile) and Phase 3 (sub-adult) stages were not affected by dietary ARA levels (P > 0.05). In Phase 3, whole-body fatty acid profiles reflected those of the diets except for eicosapentaenoic acid (EPA) content, being significantly higher in the ARA 0 group, demonstrating that rainbow trout are capable of bioconversion of α-linolenic acid to EPA. EPA and docosahexaenoic acid contents in the ovary were less affected by dietary ARA, likely reflecting the selective deposition of those fatty acids in embryonic development. Plasma superoxide dismutase, catalase, and lysozyme activity significantly increased in both pre- and post-stress groups as a result of incremental increases in dietary ARA (P < 0.05). The expression of tnf-α was significantly downregulated in ARA-M and H relative to other treatment groups suggesting an anti-inflammatory effect. The results of this study demonstrated that dietary ARA did not affect growth performance, survival, and feed utilization indices of fry, juvenile and sub-adult rainbow trout. In contrast, dietary ARA showed significant increases in non-specific immunity parameters pre- and post-stress and downregulation of tnf-α, indicating a role in modulating fish health.