High interleukin (IL)-1β gene expression was observed in dead giant grouper brains after nervous necrosis virus (NNV) infection. To investigate the neuronal death caused by NNV infection, primary tissue culture of giant grouper brains (pGB) was performed. In NNV-infected pGB cells, the viral capsid protein was detected in both neurons and microglia; furthermore, microglial proliferation and neuronal death were observed. The culture supernatant (CS) of NNV-infected pGB cells contained IL-1β and tumor necrosis factor-α, which were mainly released from the microglia. A new batch of pGB cells was treated with CS, resulting in neuronal death, which could be prevented by blocking the IL-1β in the CS by using anti-IL-1β polyclonal antibodies. Moreover, pGB cells treated with recombinant IL-1β showed microglial proliferation and neuronal death. Thus, NNV infection may activate microglial proliferation and stimulate microglial secretion of IL-1β, which is a critical cytokine responsible for neuronal death in NNV-infected grouper brains.