The epithelium is a first line of defense against microorganisms in the gut. Reactive oxygen species (ROS) have an important role in controlling the normal gut microbiota and pathogenic bacteria. Dual oxidase 2 (DUOX2) is an important source of hydrogen peroxide in the small and large intestine, and the gut microbiota induces Duox2 expression. Here, we investigated the microbial regulation of Duox2 expression. We found that Duox2 was expressed by intestinal epithelial cells mainly in the tip of the epithelium. Duox2 expression was strongly induced by the presence of a normal microbiota in mice, but not when germ-free mice were colonized with various commensal bacteria. Duox2 expression was more rapidly induced by the gut microbiota in the colon than in the ileum. Furthermore, we showed that regulation of Duox2 expression in the ileum involved TIR-domain-containing adaptor protein including interferon-β (TRIF) and canonical nuclear factor-κB p50/p65 signaling, whereas regulation of Duox2 expression in the colon involved MyD88 and the p38 pathway. Collectively, these data indicate that the gut microbiota uses two distinct signaling pathways to induce Duox2 expression in the ileum and colon epithelium.