Traditionally, montmorillonite (Mt) was used as a pelletizing aid in the production of feed pellets, or absorbents for aflatoxin B1 and heavy metal in aquaculture. The physiological metabolism influence of Mt. on aquatic animals has been long neglected. Therefore, this study aimed to investigate the influence of dietary Mt. supplementation (0%, 0.05%, 0.1%, 0.4% and 1% Mt) on the growth performance, antioxidant capacity, intestinal barrier and microbiota composition in Marsupenaeus japonicus. The results showed that although growth performance was not influenced by the dietary Mt. supplementation, shrimp fed with 0.05% Mt. significantly increased antioxidant enzymes (superoxide dismutase and glutathione in the hepatopancreas) activities and genes expression (superoxide dismutase 5, peroxiredoxin and catalase) in the hepatopancreas and intestine. Moreover, intestinal barrier function was significantly influenced, which was identified by the increased villus height and width, upregulated barrier-related genes (fascicilin II and integrin) expression. The relative abundance of intestinal probiotic (Lactobacillus, Bacteroidales S24–7 group, Ruegeria and Faecalibacterium) were increased when shrimp fed with 0.05% and 1% Mt. In addition, shrimp fed with 0.05% Mt. significantly increased the relative abundance of Alloprevotella and decreased the relative abundance of Escherichia-Shigella, while the relative abundance of Vibrio was increased when Mt. supplementation level reached at 1%. Overall, these results indicated that dietary appropriate Mt. supplementation (0.05%) could improve antioxidant capacity, intestinal barrier and modulate microbiota composition in M. japonicus. These findings might provide partial theoretical evidence for the effects of dietary Mt. supplementation on the physiological metabolism in M. japonicus.