In order to explore the response mechanism of tilapia Oreochromis to high temperature stress, transcriptome analysis was conducted in liver tissue samples collected from Nile tilapia Oreochromis niloticus cultured for 12 days exposed to 36 ℃ (high-temperature group) and to 28 ℃ (control group) for 30, 50, and 70 d by RNA-seq technology. The results showed that there was a total of 39.23 Gb of clean data in Nile tilapia in the high-temperature group and the normal temperature control group. Comparison revealed that there were 4 342 differentially expressed genes in libraries 28 ℃-30 d and 36 ℃-30 d; 3 139 differentially expressed genes in libraries 28 ℃-50 d and 36 ℃-50 d; and 3 042 differentially expressed genes in libraries 28 ℃-70 d and 36 ℃-70 d. Further, GO and KEGG analysis indicated that high temperature stress was involved in multiple biological pathways, mainly glycolysis/gluconeogenesis, cell cycle, endoplasmic reticulum protein processing signaling pathway, mTOR signaling pathway and insulin signaling pathway. Finally, 8 differential genes were verified in the mTOR signaling pathway and related pathways by RT-qPCR experiments, which confirmed the reliability of the transcriptome sequencing results. The findings indicated that genes involved in high temperature stress in the liver tissue of Nile tilapia were involved in multiple biological processes including growth, protein folding and energy metabolism, and that provided the foundation for in-depth study of the regulation mechanism of tilapia in response to high temperature stress.