Abstract: Infectious hematopoietic necrosis virus (IHNV) is one of the pathogens responsible for massive mortality in juvenile rainbow trout. Vaccines can induce immune response in the body, which leads to the production of appropriate antibodies that help protect against pathogen invasion. Therefore, the development of a safe and effective vaccine is the key for preventing IHNV outbreaks. Vaccine safety experiments revealed no clinical signs of IHN or mortality in rainbow trout, proving that the IHNV viral fluid was completely inactivated. The experiment was divided into control, vaccine, and coumarin+vaccine groups. The vaccine was injected in a dose of 150 μL, and the control group was injected with the same dose of phosphate buffer solution (PBS). The IHNV attack experiments were performed 14d post-vaccination (D14), and samples of rainbow trout spleen and serum were taken at D7, D14, D15, D21, and D30. The IHNV attack showed a survival rate of 66% in the coumarin+vaccine group compared to 62% in the inactivated vaccine group and 24% in the control group. Histopathological analysis showed minimal splenic tissue damage and mild inflammatory reaction in the coumarin+vaccine group after IHNV tapping, whereas the control group showed severe melanin macrophage center (MMC) and tissue inflammation (TI). In addition, at D7—D30, the serum levels of total superoxide dismutase (T-SOD), catalase (CAT), alkaline phosphatase (AKP), and acid phosphatase (ACP) activities in the coumarin+vaccine group were significantly higher than those in the vaccine and control groups (P<0.05). At D7—D14, the serum levels of ghrelin transaminase (GPT) and ghrelin oxaloacetate transaminase (GOT) were significantly elevated in the coumarin+vaccine group, while after IHNV attack (D15—D30), the levels of GPT and GOT in coumarin+vaccine group were significantly lower than those in the control group (P<0.05). Gene expression analysis showed a significant upregulation of MHC-I, MHC-II, and IFN-γ in the spleen in the coumarin+vaccine group after vaccination and IHNV challenge (P<0.05). Meanwhile, the expression of CD4, CD8, IgT, C3, VIG1, and Mx1 genes was significantly higher in the coumarin+vaccine group than that in the vaccine and control groups (P<0.05). These results suggest that an inactivated vaccine combined with the adjuvant coumarin is an effective method for preventing IHNV infections, which may help to further control IHNV outbreaks in rainbow trout farming.