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Polyamine concentrations in the intestine are regulated by their biosynthesis by hundreds of gut microbial species and these polyamines are involved in host health and disease. However, polyamine biosynthesis has not been sufficiently analyzed in major members of the human gut microbiota, possibly owing to a lack of gene manipulation systems. In this study, we successfully performed markerless gene deletion in Bacteroides dorei, one of the major members of the human gut microbiota. The combination of a thymidine kinase gene (tdk) deletion mutant and a counter-selection marker tdk, which has been applied in other Bacteroides species, was used for the markerless gene deletion. Deletion of tdk in B. dorei caused 5-fluoro-2′-deoxyuridine resistance, suggesting the utility of B. dorei Δtdk as the host for future markerless gene deletions. Compared to parental strains, an arginine decarboxylase gene (speA) deletion mutant generated in this system showed a severe growth defect and decreased concentration of spermidine in the cells and culture supernatant. Collectively, our results indicate the accessibility of gene deletion and the important role of speA in polyamine biosynthesis in B. dorei.