Infectious hematopoietic necrosis (IHN) caused by the viral pathogen infectious hematopoietic necrosis virus (IHNV) is a highly contagious disease of salmonid species, resulting in significant economic impact. The previous study showed a hydroxycoumarin derivative 7-[6-(2-methylimidazole) hexyloxy] coumarin (D5) significantly inhibited spring viremia of carp virus (SVCV) infection, suggesting that D5 may be useful as a potential anti-IHNV agent. In this study, D5 at the concentration of up to 10 mg/L significantly inhibited IHNV replication in epithelioma papulosum cyprini (EPC) cells with a maximum inhibitory rate of >90%, maintained mitochondrial membrane potential (ΔΨm) levels, and decreased IHNV-induced apoptosis in virus-infected cells. As the consequence of protection on mitochondria, D5 enhanced antioxidant enzyme activities and decreased reactive oxygen species (ROS) to maintain the antioxidant-oxidant balance of IHNV-infected EPC cells. For in vivo study, D5 via intraperitoneal injection exhibited an anti-IHNV effect in the virus-infected fish by substantially enhancing the survival rate. Meanwhile, up-regulation of six interferon (IFN) related gene expressions demonstrated that D5 may activate IFN-related expressions for inhibiting IHNV replication during the early stage of viral infection, which is beneficial for the continuous antiviral action on controlling low viral loads in rainbow trout juvenile. Thus, D5 effective regulated IHNV-induced undesirable conditions to be an excellent potential therapeutic agent against IHNV infection.