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The present study was conducted to investigate the effects of glutamine (Gln) supplementation on intestinal inflammatory reaction and mucosa barrier of broilers administrated with lipopolysaccharide (LPS) stimuli. A total of 120 1-d-old male broilers were randomly divided into four treatments in a 2 × 2 experimental arrangement, containing immune challenge (injected with LPS in a dose of 0 or 500 μg/kg of body weight) and dietary treatments (supplemented with 1.22% alanine or 1% Gln). The results showed that growth performance of broilers intra-abdominally injected with LPS was impaired, and Gln administration alleviated the adverse effects on growth performance induced by LPS challenge. Furthermore, Gln supplementation reduced the increased concentration of circulating tumor necrosis factor-α, interleukin-6 and interleukin-1β induced by LPS challenge. Meanwhile, D-lactic acid and diamine oxidase concentration in plasma were also decreased by Gln supplementation. In addition, the shorter villus height, deeper crypt depth and the lower ratio of villus height to crypt depth of duodenum, jejunum and ileum induced by LPS stimulation were reversed by Gln supplementation. Gln administration beneficially increased LPS-induced reduction in the expression of intestine tight junction proteins such as zonula occludens protein 1 (<i>ZO-1</i>), <i>claudin-1</i> and <i>occludin</i> except for the <i>ZO-1</i> in duodenum and <i>occludin</i> in ileum. Moreover, Gln supplementation downregulated the mRNA expression of <i>toll-like receptor 4</i>, <i>focal adhesion kinase</i>, <i>myeloid differentiation factor 88</i> and <i>IL-1R-associated kinase 4</i> in <i>TLR4/FAK/MyD88</i> signaling pathway. Therefore, it can be concluded that Gln administration could attenuate LPS-induced inflammatory responses and improve intestinal barrier damage of LPS-challenged broilers.