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Heat stress (HS) changes the rumen fermentation of dairy cows hence and affects the metabolism of rumen papillae, the potential destruction of ruminal epithelial barrier may cause the lipopolysaccharide produced in rumen enter the inner environment, which contribution to reduction in milk performance. The aim of the present study was to pursue whether the barrier function of ruminal epithelial was damaged during heat stress. 8 multiparous Holstein dairy cows (238±10 DIM; 618±100kg of BW; 23±2.8kg of milk/d) with rumen cannula were randomly equally allocated to 2 batches, the trial was divided into 2 periods, in each period, one batch was randomly selected and allocated to 4 individual environmental chambers, and the control phase as well as trial phase were contained in each period whereas each phase is 9d. In control phase, 4 cows in one batch were in thermal neutral conditions and fed ad libitum. While in trial phase, cows in one batch were randomly divided into 2 groups. Group 1 was exposed to cyclical HS conditions and fed ad libitum, whereas cows in group 2 were pair-fed to group 1 and remained in TN conditions. Compared with PFTN conditions, HS decreased milk yield (34.22%) and milk protein (10.44%), increased the content of total VFA (31.60%) as well as its component including acetate (39.51%), propionate (47.29%) and valerate (61.54%), meanwhile HS raised the parameter of respiratory rate (RR; 2.65-fold), rectal temperature (RT; 1.33°C) and skin temperature (ST; 4.59°C). Results of micromorphological observation showed that HS caused significant proliferation of corneum and separation of layers in ruminal epithelium, meanwhile increased the thickness of corneum and granulosum. KEGG analysis for differently expressed genes(DEGs) in rumen papillae (238 up-regulated and 263 down- DEGs, Padj ≤ 0.05) showed that pathways associated with DNA replication as well as repair and amino acid metabolism were activated by HS. Results from DAVID and ClueGo indicated that biological processes related to sister chromatid segregation, Double-strand break repair, Meiotic nuclear division and etc. also up-regulated by HS, meanwhile the MAPK and NF-kB cell signaling pathway were inhibited by HS in rumen papillae. In addition, the expression of tight junction related genes was indifferent between two groups. These data suggest that HS directly aggravates and damages the physical barrier of the ruminal epithelium to some extents but doesn’t break the tight junctions between cells . A mechanism may maintain the barrier of rumen papillae under HS, during which the synthesis of milk protein was affected.