Recent studies have revealed the beneficial effects of stachyose on intestinal histology and digestive function of fish. However, a comprehensive understanding of stachyose's impact on intestinal health of fish remains unclear, limiting its use in aqua-feed. In the present study, a 12-week feeding trial was conducted to investigate the effects of dietary stachyose on intestinal microbiota and mucosal barrier function of turbot (S. maximus L). Three isonitrogenous and isolipidic experimental diets were formulated to contain 0%, 1.25% and 5% stachyose, respectively. Sequencing of bacterial 16s rRNA V-4 region indicated that dietary stachyose altered the intestinal adherent microbiota profile, which was supported by the diet-cluster of PCA, PCoA, beta diversity heatmap and phylogenetic tree. LEfSe and MetaStat analysis indicated that both 1.25% and 5% dietary stachyose significantly elevated the abundance of intestinal cellulose-degrading bacteria. However, the higher level of stachyose (5%) increased the abundance of intestinal beneficial bacteria as well as that of potential pathogenic bacteria. Moreover, 1.25% dietary stachyose significantly up-regulated the genes expression of occludin, claudin-3, and ZO-1, and down-regulated the gene expression of claudin-like in the intestine (P<0.05). Dietary stachyose at 5% significantly increased mucin-2 secretion and the gene expression of ZO-1, while significantly decreased the gene expression of claudin-like in the intestine (P<0.05). Collectively, our study showed that dietary stachyose supplementation could favorably modulate the profile of intestinal microbiota and enhance the intestinal mucosal barrier function in juvenile turbot. Stachyose showed promising potential of being used as prebiotic in diet for enhancing the intestinal health of turbot.