High-mobility group box 1 (HMGB1), a highly conserved DNA-binding protein, was involved in nucleosome formation and transcriptional regulation, and could also act as an extracellular cytokine to trigger inflammation and immune responses. In this study, we identified a HMGB1 gene in turbot (Scophthalmus maximus L.). The full-length SaHMGB1 cDNA includes an open reading frame of 615 bp which encoded a 204 amino acid polypeptide with an estimated molecular mass of 23.19 kDa. SaHMGB1 was closely related to several fish HMGB1 and shared 74.4% overall identity with human. In addition, phylogenetic analyses revealed SaHMGB1 showed the closest relationship to Larimichthys crocea. Furthermore, QPCR analysis showed that SaHMGB1 was expressed in all examined tissues with abundant expression levels in brain, gill, intestine, and head kidney, and showed different expression patterns following different bacterial challenge. The significant quick regulation of SaHMGB1 in mucosal surfaces against infection suggest that HMGB1 might play critical roles in mucosal immunity against bacterial challenge. Finally, the in vitro binding assay showed that SaHMGB1 had strong binding ability to LPS, LTA, and PGN. Functional studies should further characterize HMGB1 function to understand the importance of the integrity of the mucosal barriers against infection, and to facilitate selection of the disease resistant family/strain in turbot.