Doenjang has been reported to exhibit antioxidant, fibrinolytic, antimutagenic, anticancer, and antiobesity effects. In our preliminary study, doenjang decreased fecal lipopolysaccharide (LPS) levels in mice. Therefore, we investigated the effect of doenjang on the composition of gut microbiota in mice. Treatment with doenjang significantly increased the number of bifidobacteria cultured in BL media, compared with mice not treated with doenjang. However, doenjang decreased the number of Enterobacteriaceae cultured in DHL media. Doenjang significantly suppressed the β-glucuronidase activity, but did not influence α-/β-glucosaminidase and α-/β-glucosidase activities. When gut microbiota in mice treated with or without doenjang was analyzed by pyrosequencing, doenjang induced a significant modulation of the populations of the dominant gut microbiota. At the phylum level, doenjang treatment resulted in a significant decrease of Firmicutes and an increase of Bacteroidetes, which led to a decrease in the Firmicutes to Bacteroidetes ratio in gut microbiota. At the family level, the number of Ruminococcaceae and Lachnospiraceae were significantly decreased, while the number of Odoribacter_f was increased in doenjang-treated mice. Of colonic tight junction proteins, occludin, ZO-1, and claudin-1 in mice, occludin alone was significantly increased by treatment with doenjang. Although treatment with doenjang seemed to suppress NF-κB activation, it was not significant. Doenjang significantly suppressed tumor necrosis factor-α expression, whereas it did not influence interleukin (IL)-1β and IL-6 expression. However, doenjang increased IL-10 expression. Based on these findings, doenjang may promote gut health by regulating gut microbiota and its LPS concentrations and suppressing harmful enzyme production.