The aim of this study is to investigate the protective effect and regulatory mechanism of Gln on the enteropathy of turbot induced by soybean meal. Three isonitrogenous and isolipidic practical diets were formulated with fish meal basis (FM), soy protein replacing 40% fish meal protein (SBM) and dietary 2% Ala-Gln in SBM (Ala-Gin) for sextuple replicates of 30 fish each tank for 12 weeks. The fish in SBM group presented distinct enteropathy including declined absorptive surface and overt infiltration of mixed leukocytes in the lamina propria. However, the turbot fed Ala-Gln diet showed the integrity of the intestinal tissue structure and higher ADC and AKP levels like fish in FM group. Compared to SBM group, the inclusion of 2% Ala-Gln in SBM-basal diet significantly elevated the expression of barrier-forming tight junction proteins claudin-4, occludin and ZO-1 and depressed the expression of pro-inflammatory cytokines TNF-alpha in the intestine. Fish fed Ala-Gln diet showed remarkable increased MUC-2 and PPAR-gamma gene expression as well as reduced expression of NF-kappa B and MLCK equivalent to the level of FM group. Additionally, Gln significantly elevated the relative abundance of Proteobacteria (Vibrio spp.) and declined Bacteroidetes (Bacteroides spp.) relative abundance. The similarity of intestinal microbial communities in Ala-Gln group was notably closer to FM group from PCoA, UPGMA and Heatmap analyses based on weighted UniFrac distance. In conclusion, dietary Gln could probably enhance intestinal barrier function to alleviate enteropathy by improving MUC-2 and PPAR-gamma expression and inhibiting NF-kappa B-MLCK signaling pathway, as well as altering microbiota.