Selenopeptides may be a valuable bioactive compound to promote gut microbiota-targeted therapeutic methods for intestinal disease and hepatopathy. However, limited information is available on the utilization of selenopeptides by gut microbiota, especially Selenium (Se) function. For this purpose, the present study aimed to investigate the protective effect of selenopeptide (RYNA(Se)MNDYT, Se-P2, purity of ≥ 95%) and its original peptide (RYNAMNDYT, P2, purity of ≥ 95%) in vivo by the microbiota-metabolite axis and further analyze the potential contribution of Se biofortification to Se-P2 bioactivity. The results showed that Se-P2 exhibits a higher protective effect on lipopolysaccharide (LPS)-induced inflammation than P2, including pathology of the colon and liver, which suggested that the bioactivity of P2 was promoted by the organic combination of Se. Notably, gut microbiota composition tended to be a healthy structure by Se-P2 pretreatment in LPS-injured mice, which had a positive effect on LPS-induced gut microbiota dysbacteriosis. Additionally, only Se-P2 promoted an increase in the relative abundance of Lactobacillus, Alistipes, and Roseburia and a decrease in the relative abundance of Akkermansia, Erysipelatoclostridium, and Bacteroides in LPS-injured mice. The changes in gut microbiota were obviously correlated with the changes in metabolites and affected the metabolic pathways of valine, leucine, isoleucine, phenylalanine, tyrosine, and tryptophan biosynthesis and phenylalanine metabolism. This may be one of the key reasons for Se-P2 to exert bioactivity through the microbiota-metabolite axis. Furthermore, Se-biofortification in Se-enriched Cordyceps militaris affected the parental proteins of Se-P2 to modulate mitogen-activated protein kinase, GPI anchored protein, and carbohydrate metabolism, translation, folding, sorting and degradation, which may contribute to the bioactivity of Se-P2. Our study provides information on the effect of Se on selenopeptides in vivo, which further promotes the prospective applications of selenopeptides as dietary supplements.