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The growth retardation of yaks commonly exists on the Tibetan Plateau, and the gastrointestinal barrier function of growth-retarded yaks is disrupted. Glutamine (Gln) is an effective feed additive to improve the gastrointestinal barrier function of animals. This research evaluated the effects of Gln on growth performance, serum permeability parameters, gastrointestinal morphology and barrier function of growth-retarded yaks. Thirty-two male growth-retarded yaks (74.0 ± 6.16 kg of BW and 480 ± 5.50 days of age) were randomly allocated to 4 groups: the negative control (GRY, fed basal ration), Gln1 (fed basal ration and 60 g/d Gln per yak), Gln2 (120 g/d) and Gln3 (180 g/d). Another 8 male growth normal yaks (112 ± 6.11 kg of BW and 480 ± 5.00 days of age) with same breed were used as a positive control (GNY, fed basal ration). The results showed that GRY had lower growth performance and higher (P < 0.05) diamine oxidase, D-lactic acid and lipopolysaccharide concentrations in serum as compared to GNY. Glutamine improved the average daily gain (ADG) of growth-retarded yaks, and the Gln2 group displayed highest ADG. Glutamine supplementation reduced markers of gut permeability in growth-retarded yaks. The GRY and Gln2 groups were selected to study the gastrointestinal barrier function. Growth-retarded yaks fed Gln2 showed higher (P < 0.05) height and surface area of ruminal papillae as compared to GRY. A similar trend of height and surface area in jejunal villus was found between GRY and Gln2 groups. The Gln2 increased (P < 0.05) the concentrations of secretory immunoglobulin A in jejunum and ileum of growth-retarded yaks. The rumen and jejunum of Gln2 yaks exhibited lower (P < 0.05) interleukin-1β and higher (P < 0.05) interleukin-10 mRNA expressions. Growth-retarded yaks fed Gln2 increased (P < 0.05) the expressions of claudin-1, occludin and zonula occludens-1 in the rumen and jejunum. In conclusion, dietary supplementation with Gln could improve the gastrointestinal barrier function and promote the compensatory growth of growth-retarded yaks., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)