Alternative oil sources are needed to meet the growing demand for highly digestible sources of energy and fatty acids in fish feeds. Historically, marine oils have met this need; however, diminishing supplies cannot continue to meet the demand of a rapidly growing aquaculture industry. Furthermore, the primary dietary source of long-chain polyunsaturated fatty acids for humans is seafood, but for farmed fish to meet the dietary LC-PUFA requirements of human consumers, aquafeeds must contain oil sources high in these fatty acids, such as fish oil (FO). The aim of this study was to evaluate the effects of Latitude™ oil (Transgenic canola) inclusion in fish feeds on growth performance, non-specific immune responses, histology, and fillet omega-3 fatty acid contents in rainbow trout, Oncorhynchus mykiss, fed for 52 weeks. Latitude oil (LO) is highly digestible (93%), containing omega-3 fatty acids eicosapentaenoic acid (EPA, C20:5n-3) docosapentaenoic acid (DPA, C22:5n-3) and docosahexaenoic acid (DHA, C22:6n-3). Three isonitrogenous (49.8%), isolipidic (20.4%) and isocaloric (24.2 MJ/kg) diets differing by lipid source (0, 8, or 16% LO, replacing FO and poultry fat) were fed over an entire production cycle beginning with 19g juvenile fish. At the end of 52-week feeding trial, final body weight, weight gain and specific growth rate of fish fed 8% LO (LO-8) and 16% LO (LO-16) diets were significantly higher than those fed the 0% LO (LO-0) diet (P < 0.05). Phagocytic respiratory burst in fish fed the LO-16 diet was significantly higher than those fish fed the other 2 diets (P < 0.05). There were no significant differences in superoxide dismutase (P=0.295), catalase (P=0.078) and lysozyme (P=0.075) activities among different dietary groups. Histological examination of the distal intestine indicated reduced inflammation in fish fed the LO-8 diet but not the LO-0 and LO-16 diets. Inclusion of LO enhanced the omega-3 fatty acid concentrations of EPA and docosahexaenoic acid (DHA, 22:6n-3) in the fillet. Fillet DHA content of fish fed the LO-8 and LO-16 diets were similar to those of fish fed the LO-0 diet. As these diets had lower DHA content, this suggests dietary EPA and DPA from LO was converted to DHA and deposited in the fillet. This is supported by increased expression of genes involved in fatty acid elongation, desaturation and beta oxidation in both liver and muscle of fish fed LO (P < 0.05). Total EPA+DHA content of the edible fillet ranged between 1079 to 1241mg/100g across treatments, each providing the recommended daily intake for human consumption. Overall, this study demonstrated that LO is a highly digestible lipid source suitable for meeting the fatty acid requirements of rainbow trout, as well as consumer expectations for fillet omega-3 fatty acid content.