Nano‑selenium (nano-Se) shows high biological activity and low toxicity, and has emerged as an ideal antioxidant. Our goal was to determine the underlying mechanism of nano-Se-mediated heat stress tolerance in rainbow trout (Oncorhynchus mykiss). Using liquid chromatography-mass spectrometry (LC-MS) metabolomics, histomorphology, and conventional biochemical assays, we investigated the physiological responses of heat-stressed rainbow trout to nano-Se. Fish were fed to two levels nano-Se at 18 °C for 9 days: CG18 (0 mg/kg) and Se18 (5 mg/kg). The water temperature of all groups was increased to 24 °C and maintained for 8 h (CG24, Se24). The results showed that most glycerophospholipids and CoA levels were decreased in CG18-CG24, and pathway enrichment analysis showed that it mainly interfered with glycerophospholipids and fatty acid metabolism. Meanwhile, hematoxylin and eosin and Oil Red O staining showed significant damage to CG18-CG24, which was ameliorated by Se18-Se24. The results combining analysis of antioxidant enzymes and heat shock proteins further support the notion that nano-Se supplementation inhibited galactose metabolism and activated the glutamate-glutamine metabolic pathway as the key metabolic strategy against heat stress. These results could establish heat stress defense strategies and increase our understanding of the mechanism of nutrient participation in fish's response to adverse environments. SIGNIFICANCE: Global warming is affecting the distribution and survival of cold-water fish worldwide, through seasonal water temperature increases and an increase in the frequency of extreme heat wave events. Surprisingly, Nano‑selenium (Nano-Se) with its outstanding advantages of high biological activity and low toxicity, making it a good Se nutrient supplement and free radical scavenger, and also an ideal and ecological way to supplement Se. How to utilize the metabolome to better address the complexity of the interactions that may occur with Nano-Se during the process of heat stress resistance is an important challenge. In the present study, this is the first publicly available metabonomics study of the anti-heat stress effect of Nano-Se as a nutrient on rainbow trout liver. These data indicated that Nano-Se effectively alleviated stress damage in rainbow trout, in which heat stress interfered with the metabolism of glycerophospholipids and fatty acids significantly, causing liver cell membrane damage and lipid metabolism disorders in rainbow trout. Meanwhile, supplementation of Nano-Se downregulated galactose metabolism and activated glutamate and glutamine metabolic pathways, which seems to be a key metabolic strategy to combat heat stress. The results provide a scientific basis for the development of an anti-heat-stress feed for rainbow trout that help maintain their health, productivity and welfare under unfavorable heat conditions.