Our previous studies demonstrated that the nonstructural NV protein of infectious hematopoietic necrosis virus (IHNV) was essential for efficient viral replication and pathogenicity, and that the amino acid residues 32EGDL35 of the NV protein were responsible for nuclear localization, and played important roles in suppressing IFN and inhibiting NF-κB activity. However, little is known about the influence of 32EGDL35 on IHNV replication and pathogenicity. In the present study, two recombinant IHNV strains with deletions of NV 32EGDL35 were generated and the effect on IHNV replication and pathogenicity was explored. Our results showed that both mutants stably replicated in Chinook salmon embryo cells for 15 consecutive passages, and had similar host-tropism as wild-type (wt) IHNV; however, titers of the mutants were lower than those of wt IHNV in CHSE-214cells. Infection of rainbow trout showed wt IHNV produced 90% cumulative mortality, while the mutants produced 55% and 60% cumulative mortality, respectively. Histopathological evaluation showed that tissues from the liver, brain, kidney, and heart of fish infected with wt IHNV exhibited pathological changes, but significant lesions were found only in the liver and heart of fish infected with the recombinant viruses. In addition, the recombinant viruses induced higher expression levels of IFN1, Mx-1, and IL-6 compared with those induced by wt IHNV. These results indicated that the 32EGDL35 residues were essential for the efficient anti-IFN and NF-κB-inhibiting activity of NV. Our results provide a basis for understanding the roles of 32EGDL35 in IHNV replication and pathogenicity, and may prove beneficial in the prevention and control of IHNV infections of fish.