In this study, we investigated the black soldier fly (Hermetia illucens) larvae oil (IO) as replacement of fish oil and vegetable oil in the diets of European catfish. The formulated diets included a control diet (CTR) containing fish oil (FO) and rapeseed oil (RO), and two experimental diets replacing FO and RO, with 50% and 100% IO, respectively. The nutritional trial comprising 630 European catfish juveniles (average initial body weight: 28.1 ± 0.17 g) randomly distributed into triplicated dietary groups (CTR, IO50, and IO100) was conducted for 8 weeks in a recirculation aquaculture system. The study was focused to evaluate the impact of IO on fish growth and nutrient utilisation, lipid metabolism, antioxidant, and immune-related responses in juvenile fish. Results obtained indicated that all the diets promoted adequate fish growth and feed utilisation, with no significant differences in these parameters between the dietary fish groups. It appears that the diets did not impact the activity of digestive enzymes, but a decrease in the apparent digestibility coefficients was observed. Significant differences were observed in tissue fatty acid (FA) composition. The significantly higher levels of whole-body total saturated fatty acids and arachidonic acid in fish fed IO-based diets relative to those fed CTR diet were accompanied by significantly lower levels of whole-body monounsaturated fatty acids and n-3 polyunsaturated fatty acids (n-3 PUFA). However, some deviations in this trend were observed in the liver highlighting the sparing effect for long chain polyunsaturated fatty acids (LC-PUFAs). Gene expression analysis showed a significant upregulation of genes encoding lipoprotein lipase (lpl), carnitine palmitoyl transferase (cpt1a), and hepatocyte nuclear factor 4 alpha (hnf4α) in the livers of fish fed the IO100 diet. Among the five immune-related genes examined in the spleen, only interferon regulatory factor 1 was significantly upregulated in fish fed IO50 relative to the CTR. These results indicated upregulated metabolic activities in fish fed the IO100 diet probably due to a higher level of dietary medium chain saturated fatty acids (MCSFA) and a lower level of LC-PUFA, creating the need for fish to oxidise more MCSFA and to deposit more LC-PUFA in the tissues in order to maintain fatty acid balance.