Background: The compromised performance of laying hens in the late phase of production relative to the peak production was thought to be associated with the impairment of intestinal functionality, which plays essential roles in contributing to their overall health and production performance. In the present study, RNA sequencing was used to investigate differences in the expression profile of intestinal functionality-related genes and associated pathways between laying hens in the late phase and peak phase of production. Results : A total of 104 upregulated genes with 190 downregulated genes were identified in the ileum (the distal small intestine) of laying hens in the late phase of production compared to those at peak production. These upregulated genes were found to be enriched in little KEGG pathway, however, the downregulated genes were enriched in the pathways of PPAR signaling pathway, oxidative phosphorylation and glutathione metabolism. Besides, these downregulated genes were mapped to several GO clusters in relation to lipid metabolism, electron transport of respiratory chain, and oxidation resistance. Similarly, there were lower activities of total superoxide dismutase, glutathione S-transferase and Na + /K + -ATPase, and reductions of total antioxidant capacity and ATP level, along with an elevation in malondialdehyde content in the ileum of laying hens in the late phase of production as compared with those at peak production. C onclusion s: The intestine of laying hens in the late phase of production were predominantly characterized by a disorder of lipid metabolism, concurrent with impairments of energy production and antioxidant property. This study uncovers the mechanism underlying differences between the intestinal functionality of laying hens in the late phase and peak phase of production, thereby providing potential targets for the genetic control or dietary modulation of intestinal hypofunction of laying hens in the late phase of production.