Abstract: Rainbow trout (Oncorhynchus mykiss) is a cold-water fish widely cultivated in the world. Heat stress has a great impact on its growth and reproduction. At present, global warming and extreme high temperature in summer have seriously affected its growth and survival. To characterise their metabolic changes under heat stress, this study explored the metabolic physiological responses of rainbow trout gill target organs under high temperature exposure (20℃and 24℃) and subsequent return to initial temperature (14℃) based on UPLC-Q-TOF/MS metabolomics techniques. The results showed that compared to the control group, 128, 130 and 108 differentially significant metabolites were identified in the high temperature groups (20℃ and 24℃) and the recovery group(14℃), respectively. Metabolites related to lipid metabolism such as linoleic acid and arachidonic acid, as well as metabolites related to cellular redox status such as reduced coenzyme Ⅱ (NADPH), glutathione (GSH) and glutathione disulfide (GSSG) were significantly altered by high temperature exposure. Enrichment analysis showed that these metabolites were mainly involved in metabolic pathways such as glycerophospholipid metabolism, sphingolipid metabolism, linoleic acid metabolism, arachidonic acid metabolism, pentose phosphate pathway and glutathione metabolism in rainbow trout gills. Notably, none of the metabolic pathways, except sphingolipid metabolism, returned to normal after the temperature returned to initial. The above results suggested that heat stress led to disruption of lipid metabolism in rainbow trout gill target organs, which may lead to structural and functional damage to gill cell membranes, induced an inflammatory response in gill cells and generated an immune response. At the same time, rainbow trout gill cells regulated the GSH/GSSG ratio in glutathione metabolism through NADPH production by the pentose phosphate pathway to increase the antioxidant capacity of cells to counter oxidative stress and prevent apoptosis. The results of this study provided a scientific basis for understanding the physiological responses of rainbow trout under heat stress.