Alternative sources of fish oil (FO) are one of the major problems in aquaculture; therefore, the goal of the present study was to examine insect (black soldier fly larvae) oil (BSLO) as a potential replacer of fish/soy oil in juvenile rainbow trout (initial average weight of 32 ± 0.15 g) feed. Four diets were formulated wherein FO (control diet) was completely replaced with either soybean oil (SO) or BSLO, and an additional BSLO-based diet supplemented with 1.5% bile acid (BSLO + BA) were fed to the fish for 10 weeks. Growth performance of the BSLO fed group was similar (
P
> 0.05) to that of the FO and SO fed groups, however, the fish fed BSLO + BA diet registered the lowest growth (
P
< 0.05). Oil sources did not (
P
> 0.05) affect the major nutrient content of whole-body, however, the fatty acid composition of the muscle and liver was influenced (
P
< 0.05), with the highest 14:0, 16:0, and total saturated fatty acid detected in BSLO or BSLO + BA fed trout compared to the others (
P
< 0.001). No significant differences were observed in eicosapentaenoic acid + docosahexaenoic acid (EPA + DHA) or total n-3 polyunsaturated fatty acid (PUFA) content in muscle among the groups, whereas, the highest EPA:DHA and n-3:n-6 ratios were detected in the FO group. Gene expression for fatty acid binding protein (
fabp
)
,
fatty acid synthase (
fas
), and Δ5 desaturase in the liver was lower in FO (
P
< 0.05), while BSLO + BA registered the highest Δ6 expression (
P
= 0.006). Supplementation of BA in the BSLO diet increased superoxide dismutase (SOD) and catalase (CAT) activities compared to the other groups (
P
< 0.05). In conclusion, BSLO could serve as a substitute for FO and SO in rainbow trout diet without negatively impacting growth performance, whole-body composition and nutrient retention, and modulate the expression of fatty acid metabolism-related genes in rainbow trout.