Bisphenol A (BPA), as a kind of widely exerted environmental hazardous material, brings toxicity to both humans and animals. This study aimed to investigate the role of glutamine (Gln) in intestinal inflammation and microbiota in BPA-challenged piglets. Thirty-two piglets were randomly divided into four groups according to 2 factors including BPA (0 vs. 0.1%) and Gln (0 vs. 1%) supplemented in basal diet for a 42-day feeding experiment. The results showed BPA exposure impaired piglet growth, induced intestinal inflammation and disturbed microbiota balance. However, dietary Gln supplementation improved the growth performance, while decreasing serum pro-inflammatory cytokine levels in BPA-challenged piglets. In addition, Gln attenuated intestinal mucosal damage and inflammation by normalizing the activation of toll-like receptor 4 (TLR4)-p38/MAPK-nuclear factor-kappa B (NF-κB) pathway caused by BPA. Moreover, dietary Gln supplementation decreased the abundance of Actinobacteriota and Proteobacteria, and attenuated the decreased abundance of Roseburia, Prevotella, Romboutsia and Phascolarctobacterium and the content of short-chain fatty acids in cecum contents caused by BPA exposure. Moreover, there exerted potential relevance between the gut microbiota and pro-inflammatory cytokines and cecal short-chain fatty acids. In conclusion, Gln is critical nutrition for attenuating BPA-induced intestinal inflammation, which is partially mediated by regulating microbial balance and suppressing the TLR4/p38 MAPK/NF-κB signaling.