Simple SummaryIn this study, the transcriptomic response ofTrachinotus ovatusliver was observed against high-temperature stress. Through differential expression and short time-series expression miner (STEM) analyses, some high-temperature-related genes and biological pathways were screened, which were mainly related to protein balance, hypoxia adaptation, and energy metabolism. Our results suggest that protein dynamic balance and function, hypoxia adaptation, and energy metabolism transformation are crucial in response to acute high-temperature stress. These results contribute to understanding the molecular response mechanism ofT. ovatusunder acute heat stress and provide novel insights into the selection and breeding of heat-tolerant cultivars and the high-quality development of aquaculture.AbstractTrachinotus ovatusis a major economically important cultured marine fish in the South China Sea. However, extreme weather and increased culture density result in uncontrollable problems, such as increases in water temperature and a decline in dissolved oxygen (DO), hindering the high-quality development of aquaculture. In this study, liver transcriptional profiles ofT. ovatuswere investigated under acute high-temperature stress (31 °C and 34 °C) and normal water temperature (27 °C) using RNA sequencing (RNA-Seq) technology. Differential expression analysis and STEM analysis showed that 1347 differentially expressed genes (DEGs) and four significant profiles (profiles 0, 3, 4, and 7) were screened, respectively. Of these DEGs, some genes involved in heat shock protein (HSPs), hypoxic adaptation, and glycolysis were up-regulated, while some genes involved in the ubiquitin-proteasome system (UPS) and fatty acid metabolism were down-regulated. Our results suggest that protein dynamic balance and function, hypoxia adaptation, and energy metabolism transformation are crucial in response to acute high-temperature stress. Our findings contribute to understanding the molecular response mechanism ofT. ovatusunder acute heat stress, which may provide some reference for studying the molecular mechanisms of other fish in response to heat stress.