Intestinal ischemia, alterations in enteric flora, and pro-inflammatory cytokines (TNF-α and IFN-γ) have been shown to play key roles in the pathogenesis and recurrence of Crohn’s disease (CD) (1). Daikenchuto (TU-100), a traditional Japanese medicine (Kampo) composed of processed ginger, ginseng radix, and Japanese pepper, is a pharmaceutical grade multi-herbal medicine that is widely prescribed in Japan to improve gastrointestinal disorders such as CD (2). A randomized controlled trial on TU-100 in patients with CD is currently underway in the United States. Basic studies have demonstrated that TU-100 suppresses TNF-α and IFN-γ production in the intestinal mucosa of CD by inducing endogenous adrenomedullin (ADM) release, a potent vasodilatory peptide with anti-inflammatory activity (3,4). We have demonstrated that TU-100 contains transient receptor potential ankyrin 1 (TRPA1) channel ligands, [6]-shogaol and hydroxyl-α-sanshool, which increase intestinal blood flow via enhancing ADM release from the intestinal epithelial cells (5). This shows that TRPA1 present in the intestinal epithelial cells controls intestinal blood flow via ADM release. ADM has also been found to have an antimicrobial effect comparable to that of human β-defensin in the intestinal lumen, suggesting its potential influence on enteric flora. However, little is known regarding the effect of TU-100 on enteric microbiota, which may alter the dynamic relationship between host and microbes to ameliorate colitis. In this study, C57Bl/6 female mice (n=6, 6 weeks old) were housed together to minimize generational and environmental variables that might possibly confound the microbiota analysis. All mice were fed a defined diet (AIN-76A) supplemented with or without 15 g of TU-100 extract/kg diet (1.5% wt/wt) and studied over a 28-day period. Stool samples were analyzed for 16S rRNA gene profiles using terminal restriction fragment length polymorphism (T-RFLP) and 454 pyrosequencing. TU-100 significantly altered the T-RFLP profile of stool microbiota, but only after 28 days of TU-100 treatment. Both taxonomic assignment and operational taxanomic unit (OTU) analysis revealed an expansion of Actinobacteria and Firmicutes at the phylum level and Lactococcus sp. and Clostridium sp. at the genus level. A 28-day supplementation with TU-100 was also investigated in the dextran sulfate sodium (DSS)-induced colitis murine model. TU-100 inhibited weight loss, colon shortening and colonic histological injury, and improved disease activity index in DSS-treated mice, while inducing cytoprotective heat shock protein expression and suppressing pro-inflammatory cytokine expression, particularly TNF-α. These data address the possibility that TU-100 ameliorates DSS-induced colitis by reshaping the intestinal microbiome, paving the way for a novel therapeutic strategy for CD patients.