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Sea cucumber Apostichopus japonicus is important aquatic economic species in Asia. In recent years, due to the decline of wild resources of A. japonicus , in order to ensure the sustainable supply of seedlings, the broodstock rearing is particularly important during seedling production, reproduction is also an essential stage in the life history of A. japonicus. However，the physiology of marine animals undergoes variations during the process of reproduction. It is well known that the energy demand of broodstock increases and their immunity decreases during the reproduction. Intestinal microorganisms participate in host life activities, while the presence of pathogens and probiotics will affect host health. However, the function of intestinal microbiota in the energy and immunity during the reproduction remains unclear. In this study, we performed a metagenomic analysis of the gut microbiota in A. japonicus to investigate variations in microbial composition and KEGG pathways associated with reproductive periods. Results suggested that Firmicutes/Bacteroidetes (F/B) was gradually increasing during reproduction, which increased host energy harvest, and was difference between male and female A. japonicus. Virulence Factors of Pathogenic Bacteria (VFBD) upregulated from the gonadal growth period. The higher expression of VFBD during the gonadal growth period than at other times may be caused by the fact that Flavobacteriaceae and Rhodobacteraceae are most abundant during this period. Lysozyme activity was upregulated during reproduction and peaked during the gonadal mature period, with a similar trend of variability as in VFBD, which carried out immunological protection against A. japonicus. The relative abundance of Saccharomycetaceae , and Bacillaceae was highest during the spawning period, and it has been identified that microorganisms strengthen immune protection against A. japonicus during this period, when A. japonicus was in a weakened state. Based on KEGG analysis, expression of oxidative phosphorylation and the fever pathway increased after spawning, indicating that reproduction causes oxidative stress in A. japonicus. Additionally, the differences in several KEGG pathways were related to sex. Overall, by analyzing the energy and immune systems, the study confirmed that intestinal microorganisms play vital physiological roles in broodstock of A. japonicus during reproduction. This study provided important information for breeding and the application of A. japonicus in aquaculture. • Sea cucumber Apostichopus japonicus , is one of the most economically important aquaculture species in Asia. This study confirmed that intestinal microorganisms play vital physiological roles in broodstock of A. japonicus during reproduction, microorganisms participate regulation of host energy metabolism, immune adjustment. • There are several pathogenic bacteria increased during reproduction. Post spawning, the number of probiotics increases, which can protect A. japonicus and provide immune compensation. • The composition of intestinal microbiome is also different in male and female individuals. • Several KEGG pathways changed, focused on energy metabolism and oxidative damage. • This study provided an important information for breeding and the application in aquaculture of A. japonicus. [ABSTRACT FROM AUTHOR]