With the development of industries, human activities, particularly the burning of fossil fuels and deforestation, have worsened the natural greenhouse effect and aggravated the degree of global warming. It is estimated that the global average temperature will increase by 1–4 ℃ by the end of the century due to the continuous growth in CO2 emissions, which will simultaneously lead to inevitable increases in water temperatures. Rainbow trout (Oncorhynchus mykiss) is a commercially important cold-water fish. Due to its high dependence on ambient water temperature, temperature increase can adversely affect its growth and reproduction capacity and ultimately its health, even leading to death. Therefore, it is of great significance to understand the molecular mechanism of rainbow trout in response to heat stress. Metabolomics is a technology to analyze the disturbance of metabolic pathways in an organism by detecting the changes of endogenous metabolites after stimulation or disturbance. In combination with ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS), it enables us to identify the holistic variable metabolites and related metabolic pathways in a living system by measuring the low-molecular-weight metabolites. In this study, heat stress experiments at 20 ℃ (T20 group) and 24 ℃ (T24 group) were carried out, taking the liver as the targeted organ, to investigate the metabolic response mechanism of rainbow trout under heat stress at the cellular level. UPLC-QTOF-MS metabolomics was used to explore the changes of endogenous metabolites in rainbow trout liver and find the differential metabolites and related metabolic pathways. Overall, 65 differential metabolites were screened in the T20 group, and were significantly enriched in 17 metabolomic pathways, including linoleic acid, galactose, α-linolenic acid, glycerophospholipid, purine, sphingolipid, and glutathione metabolisms. Meanwhile, 80 differential metabolites were screened in the T24 group, and were mainly enriched in 15 metabolomic pathways, including linoleic acid, retinol, glycerophospholipid, sphingolipid, α-linolenic acid, glutathione, and glyceride metabolisms. Among them, lipid metabolism was most significantly affected, followed by amino acid metabolism. In conclusion, heat stress induced oxidative stress in rainbow trout liver, causing activation of the glutathione metabolic pathways in a short period to accelerate the scavenging of reactive oxygen species. However, continuous heat stress damages the homeostasis of lipid metabolism, causing the reduction of metabolites such as DHA and α-linolenic acid that maintain the normal function of cells, resulting in an imbalance of the body’s immune and antioxidant systems, and further causing hepatocyte injury. This study provides insights for follow-up studies regarding the heat stress regulation mechanism of specific metabolic pathways, and a solid theoretical basis for the multi-perspective exploration of the heat resistance mechanism of rainbow trout.