High mobility group box 1 (HMGB1) is a nuclear weapon in the immune arsenal and a master regulator of innate immunity, at the crossroads between innate and adaptive immunity. To clarify the immune characterizations of HMGB1 in fishes, two co-orthologs of HMGB1 (CiHMGB1a and CiHMGB1b) were identified in grass carp Ctenopharyngodon idella by local EST database searching and RACE techniques. mRNA expressions of the two HMGB1 genes are widespread in fifteen tissues investigated. The transcripts of CiHMGB1a and CiHMGB1b were significantly up-regulated and reached peak at 24 h post GCRV challenge in spleen and head kidney tissues (P < 0.05). The modulations are slow post-bacterial PAMP stimulations by contrast with those after viral PAMP or GCRV challenge. They are inhibited by bacterial PAMPs, but are enhanced by viral PAMP or virus. mRNA expression of CiHMGB1a is high and strongly modulated by nucleic acids and transcription of CiHMGB1b is low and mildly regulated by nucleic acids and capsids of GCRV. The over-expression vectors were constructed and transfected into C. idella kidney cell line to obtain stably expressing recombinant proteins. In HMGB1 over-expressed cells, mRNA expressions of IPS-1, MyD88 and Mx1 were down-regulated, whereas TRIF was found to be up-regulated and IFN-I showed no change in its expression. After GCRV challenge, the transcripts of IPS-1, MyD88 and Mx1 were up-regulated, while IFN-I showed down-regulation, and TRIF showed up-regulation after an initial phase of decline. The titer assay demonstrated no antiviral activity of HMGB1s. The results indicated mRNA expressions of HMGB1a and HMGB1b are enhanced by GCRV or viral PAMP, and are inhibited by bacterial PAMPs; HMGB1a and HMGB1b collaborate with each other and play important roles in modulating the innate immune responses, although without direct antiviral effect; the immune network triggered by HMGB1 work together in concert to maintain homeostasis.