Cost-effective fish feed production is the basis for producing affordable fish. Black soldier fly (BSF), Hermetia illucens, larval meal is a promising alternative to animal protein sources of fish feed. In our study, BSF larval meal was substituted for fishmeal in fish diets at 0%, 25%, 50%, 50%, 75%, and 100% and named as B0, B25, B50, B75, and B100. Gut microbiota, expression of immune-related genes and resistance to Lactococcus petauri were investigated in rainbow trout (Oncorhynchus mykiss) fed these diets for 90 days. It was found that the gastrointestinal tract of rainbow trout fed diets containing BSF had a higher bacterial diversity. The abundance of Firmicutes, Actinobacteria and Proteobacteria increased in parallel with the rate of replacement of fishmeal with BSF meal. Fish fed with B75 were the most resistant to L. petauri, followed by the groups fed with B100, B50 and B25. Compared to the control group, the expression of cytokines (Tgf, Il-10, Il-1β, Tnf-α and Il-8) and immune-related genes (IgM, IgT, Mhc-II and Tlr- 5) were significantly increased (p < 0.05) in all fish groups fed BSF-containing diets. Furthermore, BSF-containing diets increased immunity and cytokine-related genes in fish. Due to the immune boost, rainbow trout can be fed a diet with 50% BSF meal substitution without removing the larvae chitin or pretreating the BSF meal.