Abstract: With worsening global greenhouse effect, aquaculture faces growing high-temperature threats. Mass mortality events of farmed fish induced by chronic heat stress (CHS) have become common. Therefore, more attention should be paid to CHS. To investigate the effects of CHS on carbohydrate and lipid metabolism, as well as mitochondrial homeostasis in fish, a total of 240 juvenile Micropterus salmoides with an average body weight of (12.04±0.15) g were randomly divided into two groups, each consisting of four replicates. The fish were cultured in thermostatically controlled aquaculture tanks at (27.0±0.5) ℃ (Control group) and (33.0±0.5) ℃ (Heat group) for 8 weeks, using commercial feed. The results demonstrated that in the Heat group, the hepatic expression levels of glucose metabolism-related genes, including IRA, IRB, IRS1, PEPCK, GSK3β, AKT1, and PBP1, were significantly downregulated. Among lipid metabolism-related genes, FAS, LPL, and HSL were significant downregulated, whereas CPT-1, FFAR, ACC, and ATGL were marked upregulated. Among genes related to mitochondrial homeostasis, SIRT1 and AMPKα were significantly upregulated, while PGC-1α, ERRα, TFAM7, OAP1, DRP1, P62, LC3, and MFN1 also showed marked downregulation. In addition, the expression levels of IκBβ, P65, Iκκβ, and IL-8 were all significantly downregulated in the Heat group. Furthermore, the Heat group exhibited abnormal glycogen accumulation and lipid deposition in the liver, accompanied by mitochondrial structural damage and a significant decrease in mitochondrial density. Liver transcriptome sequencing revealed that differentially expressed genes were primarily enriched in the C-type lectin receptor signaling pathway, cytokine-cytokine receptor interaction, actin cytoskeleton regulation, carbon metabolism, and glycolysis/gluconeogenesis signaling pathway. In summary, CHS disrupts mitochondrial dynamics homeostasis, induces mitochondrial dysfunction and energy metabolic imbalance in M. salmoides. Additionally, CHS disrupts hepatic carbohydrate and lipid metabolism, leading to abnormal accumulation of glycogen and lipids in the liver. The suppression of energy metabolic homeostasis by CHS further impairs hepatic immune function in this species. The findings of this study not only provide data supporting the elucidation of CHS-induced hepatic metabolic disorders in fish but also offer insights for developing dietary strategies to mitigate such damage in fish livers.