Astragalus polysaccharide (APS) plays important roles in antibacterial, antiviral and antiparasitic activities in mammals, birds and aquatic animals. However, the relationship between non-specific immune responses and intestinal microbiota in sea cucumber (Apostichopus japonicus) after dietary APS supplementation has not been reported to date. Here, the effect of dietary APS supplementation on the non-specific immune response and intestinal microbial composition and species distribution of sea cucumber was explored. We found that although there was no significant effect on sea cucumber growth, the enzymatic activity and expression level of immune- and antioxidant-related genes changed after dietary APS supplementation. Furthermore, the intestinal microbial composition and species distribution of sea cucumber were different at the phylum and genus levels after dietary APS supplementation. The phyla Proteobacteria and Bacteroidetes were significantly different between the APS2 group and CK group. The results of PCA and PCoA analysis also showed that the APS2 group was significantly different compared to the other groups. Finally, analysis of the relationship between non-specific immune responses and the intestinal microbiota showed that the expression level of NF-κB was significantly correlated with intestinal microbiota at the genus level. This finding suggests that dietary APS supplementation might affect the non-specific immune response and intestinal microbiota of sea cucumber through the NF-κB signalling pathway; the appropriate added level was 800 mg/kg. Taken together, our results lay a foundation for further understanding the relationship between non-specific immune responses and intestinal microbial of sea cucumber. • APS could affect the expression level of immune-related genes of sea cucumber. • Dietary APS adding could regulate the intestinal microbial diversity of sea cucumber. • Proteobacteria and Bacteroidetes was significant different between APS2 and CK group. • The APS2 group was significant different to others group by PCA and PCoA analysis. • Expression level of NF-κB was closed correlated with intestinal microbiota diversity. [ABSTRACT FROM AUTHOR]