Abstract: 【Objective】Based on systems biology analysis and chicken embryo duodenal epithelial cell model，this paper explored the mechanism of heat stress-induced intestinal injury，to screen the action targets of heat stress-induced intestinal damage in poultry，and to provide theoretical support for alleviating heat stress-induced oxidative stress in enterocytes.【Method】The NCBI database was used to screen the action targets of heat stress-induced intestinal injury， and the relevant metabolism pathways were obtained by GO functional annotation and KEGG signaling pathway enrichment analysis. Chicken embryo duodenal epithelial cells were separated to construct the heat stress model at 42 ℃. And by using pathological detection，cell activity，lactate dehydrogenase（LDH）activity，antioxidant enzyme activity，malondialdehyde（MDA）content and expression of key genes（Nrf2 and HSP70），the damages of heat stress on intestinal cells were confirmed.【Result】By taking the intersection of heat stress action targets and intestinal injury action targets，a total of 23 action targets of heat stress-induced intestinal injury were obtained，including BCL2，CD4，CFTR，ERN1， FOXM1，FOXP3，HMOX1，HSF1，HSP90AA1，HSP70，HSPA4，HSPA8，JUN，MAPK1，MTOR，NFKB1，NLRP3， PLK1，RHOB，SIRT1，SUMO2，TP53 and YAP1. KEGG signaling pathway enrichment analysis found that heat stressinduced intestinal injury were associated with NOD-like receptor signaling pathways，salmonella infection，apoptosis， cellular senescence，and protein processing in the endoplasmic reticulum. GO functional annotation analysis found that heat stress-induced intestinal injury were related to cell hypoxia response，cellular response to cadmium ion，cell response to heat，and heat shock protein binding. The heat stress model of chicken embryo duodenal epithelial cells in vitro showed that heat stress could cause cells vacuolar degeneration and in severe cases cause nucleus concentrated deep staining that leading degenerative necrosis；heat stress could reduce cell activity of chicken embryo duodenal epithelial cells and increase LDH content in cell supernatant；in addition，with the prolongation of heat stress，the MDA content in the chicken embryo duodenal epithelial cells increased，while the activities of superoxide dismutase（SOD），glutathione peroxidase （GSH-Px）and catalase（CAT）activities showed a decreasing trend；heat stress resulted in down-regulation of Nrf2 gene and up-regulation of HSP70 gene in chicken embryo duodenal epithelial cells.【Conclusion】Intracellular oxidative-antioxidative imbalance is an important factor in heat stress-induced intestinal injury，and heat shock proteins are important protective mechanism against heat stress-induced injury.