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The present study was designed to investigate whether L-arginine (Arg) supplementation would influence the production performance and reproductive traits of laying hens fed low crude protein (LCP) diets. Hy-Line brown laying hens were randomly assigned to dietary treatments of control, LCP, and LCP supplemented with 0.05, 0.10, 0.15, or 0.20% Arg for 7 wk. The results showed no significant variations on the production performance, and relative organ indexes of hens. However, significant transcriptional and structural changes were evident along the HPG axis of hens. Arg supplementation linearly upregulated (P < 0.05) the gonadotropin-releasing hormone 1 (GnRH1), and gonadotropin inhibitory hormone in the hypothalamus. The pituitary growth hormone, GnRH receptor, and follicle-stimulating hormone (FSHβ) were also increased (P < 0.05). In the ovary, GnRH1, and estrogen receptor β were linearly increased by Arg, and the ovarian morphology revealed that LCP induced structural alterations which were minimally recovered by Arg supplementation. Serum insulin-like growth factor 1 (IGF-1) and nitric oxide (NO) were increased (P < 0.05) at higher levels of Arg supplementation. Therefore, supplementing high Arg (0.20%) to LCP hens influenced the ovarian morphology and modulated the gene expression of reproductive hormones in the hypothalamic-pituitary-gonadal axis of laying hens via actions that may be related to NO and IGF-1 activity. [ABSTRACT FROM AUTHOR]