Abstract: To explore the mechanism of the intestinal tract of red swamp crayfish (Procambarus clarkii) under high temperature stress close to its physiological limit, we conducted the high-temperature stress experiments at 32 ℃ and 37 ℃, and collected the intestinal tissues after 24 h and 72 h of stress, for tissue sectioning observation and transcriptomic analysis. The results show that high temperature caused significant damages to the intestinal tissue structure of crayfish, and the damage increased with the increase of stress temperature and stress time. Compared with the control group (26 ℃), 2 462 and 4 619 differentially expressed genes (DEGs) were identified under the conditions of 32 ℃ stress for 24 h and 72 h, respectively. Under the conditions of 37 ℃ stress for 24 h and 72 h, 1 825 and 7 298 DEGs were identified, respectively. KEGG enrichment analysis shows that the DEGs were significantly enriched in the endoplasmic reticulum protein processing and metabolic pathways, and the enrichment frequency of endoplasmic reticulum protein processing pathways increased when the stress temperature increased from 32 ℃ to 37 ℃ during the same stress time, which indicates that they played an important role in coping with high temperature stress. According to the GO functional annotation, DEGs were mainly enriched in the energy metabolism-related processes such as carbohydrate metabolism and protein folding.