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This study examined the prebiotic effects of xylooligosaccharides (XOS) on intestinal characteristics, gut microbiota, cecal short-chain fatty acids, plasma calcium metabolism, and immune parameters of laying hens. A total of 1,080 White Lohmann laying hens (28 wk of age) was assigned to 6 dietary treatments that included XOS at concentrations of 0, 0.01, 0.02, 0.03, 0.04, or 0.05% for 8 weeks. Each treatment had 6 replicates with 10 cages (3 birds/cage). Blood, intestinal tissues, and cecal digesta samples were collected from chickens at the end of the experiment. Villus height, crypt depth, the villus to crypt (VH: CD) ratio, and the relative length of different intestinal sections were evaluated. Additionally, the number of microorganisms and the content of short-chain fatty acids in cecal digesta samples were determined. Plasma concentrations of immunoglobulin A (IgA), immunoglobulin G, immunoglobulin M (IgM), interleukin 2 (IL-2), tumor necrosis factor-α(TNF-α), 1, 25-dihydroxyvitamin D3 (1,25(OH)2D3), calcitonin (CT), and parathyroid hormone (PTH) were also determined. The results showed that villus height and the VH: CD ratio of the jejunum were increased (linear, P < 0.01) with the increase in dietary XOS concentration, and the relative length of the jejunum (P = 0.03) was increased significantly in XOS diets. Dietary supplementation of XOS significantly increased (linear, P < 0.01) the number of Bifidobacteria in the cecum; however, total bacteria count, Lactobacillus, and Escherichia coli in the cecum were not affected by XOS supplementation. In addition, inclusion of XOS increased (linear, P < 0.01) the content of butyrate in the cecum; and the content of acetic acid showed a linear increasing trend (P = 0.053) with increasing concentration of XOS in the diets. Supplementation with XOS increased (quadratic, P < 0.05) the content of 1,25(OH)2D3 in plasma. There were no significant differences (P > 0.05) in the content of CT and PTH among dietary treatments. Furthermore, dietary XOS increased contents of IgA (linear, P < 0.05), TNF-α (linear, P < 0.05), IgM (linear, P < 0.05; quadratic, P < 0.05), and IL-2 (quadratic, P < 0.05). Taken together, it was suggested that supplemental XOS can enhance the intestinal health and immune function of laying hens by positively influencing the intestinal characteristics, gut microbiota, cecal short-chain fatty acids, and immune parameters.