The aim of this study is to study the effects of palygorskite, a new antibacterial substance, on the growth performance, digestibility, antioxidant capacity and intestinal microbiota of Litopenaeus vannamei. The juvenile shrimp with an initial weight of (0.17±0.01) g were selected and fed with diets with 0% (control), 0.1% (S1), 0.2% (S2), 0.4% (S3), and 0.8% (S4) palygorskite, respectively, as well as the antibiotic control group diet (E) containing 0.2% enrofloxacin and were cultured for 8 weeks. The weight gain of shrimp in the palygorskite treatment groups did not increase significantly, but the survival rate of shrimp in the S1 and S2 groups was significantly higher than that in the control group, and there was no significant difference from the E group. Compared with the control group, the palygorskite supplementation groups showed increases in the activities of intestinal amylase, lipase and trypsin, as well as the lipase activity in the hepatopancreas of juvenile shrimp. The serum acid phosphatase and alkaline phosphatase activities in the S2 group were significantly higher than those in the control and E groups. The hepatopancreas superoxide dismutase activity of the S3 or S4 groups was significantly higher than that of the other groups. The α diversity analysis of intestinal microbiota showed that the Chao1 index of intestinal microbial richness decreased in the S2 group, but the coverage increased significantly. Compared with the control group, the abundance regulation of some genera in the S2 group was similar to that of enrofloxacin compared with the E group. According to the difference analysis of metabolic pathways and the mapping of interspecific relationships, palygorskite can upregulate 4 pathways of taurine, glutathione, galactose and fatty acid metabolism and increase the cooperation ability among intestinal microbes. Adding 0.1%-0.2% palygorskite to the diet can significantly improve the survival rate, digestibility and hepatopancreas antioxidant capacity of L. vannamei and play a role similar to that of enrofloxacin in regulating the abundance of intestinal microbiota in shrimp.