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The aim of this study was to test the effects of dietary L-arginine (Arg) levels on protein turnover in the small intestine and the expression of genes related to protein synthesis and proteolysis of laying hens. Xinyang Black commercial laying hens (n = 864, aged 217 d) were randomly distributed to 6 treatments with 4 replicates of 36 birds. The dietary treatments were corn-corn gluten meal based diets containing 0.64, 0.86, 1.03, 1.27, 1.42 and 1.66% L-Arg, respectively. Fractional protein synthesis rate (FSR) and fractional protein gain rate (FGR) in the jejunum were the highest in the 1.27% L-Arg group. The mRNA expression of target of rapamycin (TOR), ribosomal protein S6 kinase 1 (S6K1), and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) in the duodenum reached the highest in the 1.27% L-Arg group, while the mRNA expression of 20S proteasome (20S) was the lowest in the 1.27% L-Arg group. The mRNA abundances of TOR and S6K1 in the jejunum were the highest in the 1.27% L-Arg group, while the mRNA expression of 20S was the lowest in the 1.27% L-Arg group, and the protein expression and phosphorylation levels of TOR in the 1.27% L-Arg group were higher than those in the 0.64% L-Arg group. These results indicate that the action of an appropriate level of dietary L-Arg to improve the protein synthesis of the small intestine involves up-regulating the protein expression and phosphorylation level of TOR in the jejunum accompanied by inhibiting the mRNA expression of 20S of laying hens.