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ABSTRACT: The aim of this study was to investigate the effects of low-protein diets and the sustained release of synthetic amino acids (AA) on the performance, intestinal barrier function and nitrogen excretion of laying hens. Two hundred eighty-eight 39-week-old Hyline brown laying hens of were randomly divided into 3 groups with 8 replicates per group. The crude protein level in the control group (CON) was 16%, the crude protein levels in the crystal AA supplement group (LCP-CAA) and microencapsulated AA group (LCP-MAA) were both 13%, and the AA levels in the LCP-CAA and LCP-MAA groups were consistent with that in the CON group. The experiment lasted 12 wk, and production performance was assessed weekly. The FCR and ADFI values were significantly greater for the LCP-CAA group than for the CON and LCP-MAA groups (P < 0.05). Two hours after feeding, His levels were significantly greater in the LCP-CAA group than in the LCP-MAA group (P < 0.05); 4 h after feeding, the contents of Met, Thr, Leu and Val were significantly greater in blood from the LCP-MAA group (P < 0.05); 6 h after feeding, Trp, Ile and Arg levels were significantly greater in the LCP-MAA group (P < 0.05). The chylase content significantly decreased in the duodenum of the LCP-CAA group (P < 0.05), and the chylase and trypsin were contents increased in the ileum of the LCP-MAA group (P < 0.05). In the LCP-MAA group, significantly increased mRNA expression levels of Occludin, ZO-1 in duodenum; Occludin, ZO-1, y+LAT1 in jejunum; and ZO-1 in ileum were detected at 8 and 12 weeks (P < 0.05). The fecal nitrogen content significantly decreased in the low protein diet group (P < 0.01). In conclusion, reducing dietary crude protein levels and supplementing with microencapsulated AAs can improve intestinal barrier function, promote digestive enzyme secretion, increase the expression of AA transporters, improve dietary protein utilization efficiency, and reduce nitrogen emission in laying hens.