Heat shock protein (HSP) is a ubiquitous and highly conserved stress protein, and its expression changes are associated with the health status of fish. Hsp60 is a member of the heat shock protein family with a molecular weight of about 60 ku, which is mainly distributed in mitochondria of eukaryotic cells. Hsp60 was up-regulated during stress or cell injury and recognized by pattern recognition receptors as a danger signaling molecule. Previous studies showed that cytoplasmic Hsp60 could interact with caspase-3 to increase stress tolerance and maintain cell homeostasis. However, excessive accumulation of Hsp60 can also accelerate the activation of caspase-3, induce the release of cytochrome C and promote cell apoptosis. Hsp60 can refold and repair damaged proteins under stress by ATP binding and hydrolysis. Therefore, it is very important to study the function of Hsp60 in fish during bacterial infection. Fish in natural water are threatened by environmental pollution, natural toxins and microbial infection, and bacterial infection was a common disease. Schizothorax prenanti is a unique sub-cold-water economic fish in China and an essential variety of proliferation and release in the Yangtze River’s upper reaches. S. prenanti is a special economic animal with high content of umami amino acids and good content and ratio of amino acids and fatty acids. In recent years, the techniques of artificial domestication and breeding have been relatively mature, but the specific immune system is poor. Streptococcus agalactiae is an important pathogen that harms S. prenanti and the typical clinical symptoms of infection are exophthalmic protrusion and subcutaneous abscess on the body surface. However, the early infection of S. agalactiae is not easy to detect. As an important pathogen, S. agalactiae can spread to the whole body through blood and eventually cause pathological manifestations of edema and sepsis. After entering the blood circulation, S. agalactiae can bind with serum complement C3b through polysaccharide, transforming the activated C3b into an inhibited state to avoid non-specific immune clearance of the host. Besides, S. agalactiae can also avoid phagocytosis and clearance of macrophages through polysaccharide in the capsule, thus causing damage to the visceral tissues and organs. In this study, the molecular characteristics and response to bacterial infection were investigated to explore S. prenanti Heat shock protein 60 (SpHsp60). The 2 277 bp SpHsp60 cDNA sequence was cloned using RACE technology, which encoded 575 amino acids and had relatively conserved spatial conformation with vertebrates. Then, the analysis of tissue expression pattern showed that the expression level of SpHsp60 was the highest in hepatopancreas, followed by blood, and the lowest in skin, muscle, and intestines. Furthermore, the mRNA expression of SpHsp60 in blood, hepatopancreas, trunk kidney and spleen had significant differences in 6 h after S. agalactiae infection. Compared with the control group, the mRNA expression of SpHsp60 in hepatopancreas and trunk kidney expression significantly increased from 6 h to 72 h post-infection, while those in the spleen were significantly decreased. Our results suggested that SpHsp60 could play an early warning role in bacterial infection, providing primary data for Hsp60 mRNA expression in hepatopancreas and trunk kidney as a warning signal for S. agalactiae infection in S. prenanti.