In order to understand the immune response of Schizothorax prenanti high mobility group box 1 (SpHMGB1) to Aeromonas hydrophila stress, primers were designed by the sequence obtained from the TSA database of spleen in S. prenanti, then the sequence characterization was analyzed. Finally the response situations were detected by qPCR after A.hydrophila infection in vivo and heat-killed A.hydrophila stimuli in vitro. Sequence analysis results showed that the open reading frame of SpHMGB1 was 615 bp which encoded a peptide of 204 amino acids with a predicted molecular mass of 23.5 Ku and a theoretical isoelectric point of 6.79. The peptide contained two basic HMG boxes:box A and box B, and an acidic tail. The secondary structure was mainly composed of 47.06% of α-helices and 50% of random curls. Phylogenetic tree showed that SpHMGB1 and HMGB1 from Danio rerio and Carassius auratus were clustered together among fish HMGB1 with the high identities of 90.7% and 90.4%, respectively. After A.hydrophila infection, the relative expression of SpHMGB1 was significantly up-regulated at different time in tissues. The highest relative expression of SpHMGB1 appeared in kidney at 6 h, in blood at 72 h and in spleen at 120 h. After heat-killed A. hydrophila stimuli in macrophages, the relative expression of SpHMGB1 was down-regulated within 0.5 -24 h with the lowest at 24 h. Meanwhile the relative expression of IL-1β and TNF-α showed the up-regulated tendency. qPCR results suggested that SpHMGB1 might be involved in the immune response after A. hydrophila infection in S. prenanti. This study provided a theoretical basis for further revealing the immune-modulatory mechanism of SpHMGB1 in bacterial disease of cold-water S. prenanti.