High mobility group box (HMGB) proteins, a family of chromatin-associated nuclear proteins, exert amazingly multifaceted roles in the immune system in mammals. Thus far, little is known about the nucleocytpolasmic distribution of HMGBs in teleosts. Present study systematically investigated the dynamic localization of all the six HMGB proteins in Ctenopharyngodon idella kidney (CIK) cells. Under basal condition, all HMGBs exclusively located in nucleus. Grass carp reovirus (GCRV), polyinosinic–polycytidylic potassium salt (poly(I:C)) and lipopolysaccharide (LPS) challenge evoked nuclear export of HMGBs in various degrees: GCRV challenge induced the most nuclear export of CiHMGB2b, and the most nucleocytoplasmic shuttling of CiHMGB1b was evoked by poly(I:C) and LPS. Overall, nucleocytoplasmic shuttling of CiHMGB2a and CiHMGB3b was rarely induced by those challenges. Dynamic imaging uncovered that nucleocytoplasmic relocation of CiHMGB2b induced by GCRV occurred in cells undergoing three physiological states: karyotheca rupture, apoptosis and proliferation. Western bolt examined HMGBs-EGFP fusion proteins in whole cell lysates, cytosol, nuclear fractions and culture medium. Further study demonstrated the nucleus retention of N-terminal HMG-boxes and nucleocytoplasmic distribution of C-terminal acidic tail. Comparative analyses of the dynamic relocation of full-length, truncated or chimeric HMGBs confirmed that intra-molecular interaction between HMG-boxes and C-tail domains mediated nucleocytoplasmic translocation of HMGBs. These results firstly provide overall understanding of subcellular localization of HMGBs, and also preliminary uncover the induction mechanism of nucleocytoplasmic translocation of HMGBs by GCRV challenge, which lay a foundation for the further researches on interaction among pathogens-HMGBs-pattern recognition receptors (PRRs) in innate immunity of teleosts.