Giant groupers were immunized with two dosages (Vhigh and Vlow) of inactivated nervous necrosis virus (NNV) and subsequently challenged with NNV at 4 weeks post vaccination (wpv). Several indicators were used to analyze the protective effects of the NNV vaccine. The neutralizing antibody titer of fish serum mostly corresponded to the survival rate of immunized fish in the NNV challenge test. Extravascular IgM+ cells were detected in the brains of both NNV-infected and noninfected groupers. After NNV infection, CD8α and IgM gene expression increased in the brains, indicating CD8α+ and IgM+ lymphocyte infiltration. Moreover, the NNV load was not the highest in dead grouper brains, suggesting that this load in the brain was not the key factor for the death of groupers. However, the brains of dead fish showed the highest expression of the interleukin (IL)-1β gene, a neurotoxic factor in the brain. Therefore, IL-1β overexpression is likely to be associated with the death of NNV-infected groupers.