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In the human intestinal tract, there are more than 100 trillion microorganisms classified into at least 1000 different species. The intestinal microbiota contributes to the regulation of systemic physiologic functions and the maintenance of homeostasis of the host. It has been reported that the alteration of the intestinal microbiota is involved in metabolic syndromes, including type II diabetes and dyslipidemia, inflammatory bowel disease, allergic disease, and cancer growth. It has been reported that a microbial product from Paenibacillus polymyxa AK, which was named Enzamin, ameliorated adipose inflammation with impaired adipocytokine expression and insulin resistance in db/db mice. In order to investigate the effect of Enzamin on the intestinal microbiota and inflammation induced by obesity, mice were fed with a high-fat diet and 1% Enzamin for 4 weeks. Enzamin improved the Firmicutes-to-Bacteroidetes ratio and altered the intestinal microbiota in mice fed the high-fat diet. In addition, Enzamin suppressed the decreased expression of claudin-4 and the increased serum LPS level in mice fed with the high-fat diet. Modulating the intestinal microbiota with Enzamin may cause a decrease in serum LPS level. Based on these results, Enzamin may improve inflammation and metabolic disorders by regulating the intestinal microbiota in obese mice.