ETHNOPHARMACOLOGICAL RELEVANCE: The overuse of antibiotics has resulted in Clostridium difficile infection (CDI) as a significant global public health concern. Studies have shown that imbalances in gut microbiota and metabolism play a vital role in the onset of CDI. Shengjiang Xiexin decoction (SJT), a traditional Chinese medicinal formula widely employed in the treatment of gastrointestinal ailments, demonstrates effectiveness in addressing murine CDI. However, the precise mechanistic role of SJT in CDI treatment remains uncertain, particularly regarding its impact on gut microbiota and intestinal metabolism. Thus, further investigation is imperative to shed light on these mechanisms. AIM OF THE STUDY: This study aims to thoroughly investigate the therapeutic potential of SJT in the treatment of CDI, while also examining its impact on the intricate interplay between gut microbiota and bile acid metabolism. By employing a mouse model, we aspire to uncover novel insights that could pave the way for the development of more effective strategies in combating CDI. MATERIALS AND METHODS: We developed a mouse model for CDI and assessed SJT's potential as a therapeutic agent through pharmacological analyses. Our study employed high-throughput sequencing of 16S rRNA to identify changes in gut microbiota composition and untargeted metabolomics analysis to evaluate SJT's intervention on intestinal metabolism. We also conducted targeted analysis of bile acid metabolism to examine the specific effects of SJT. Finally, the growth-inhibitory effect of SJT on C. difficile was confirmed through ex vivo cultivation of the pathogen using cecal contents, supporting its potential role in treating CDI by modulating gut microbiota and bile acid metabolism. RESULTS: In pharmacological studies, SJT was found to effectively reduce the levels of A&B toxins and alleviate colonic inflammation in CDI mice. Mechanistically, SJT demonstrated a mild increase in the abundance and diversity of the gut microbiota. However, its most significant impact was observed in the substantial improvement of the structural composition of the gut microbiota. Specifically, SJT decreased the abundance of gut Polymorphs and Firmicutes while restoring the proportions of family Trichophyton and Bacteroides_S24-7 spp (P < 0.001). Moreover, SJT not only decreased the levels of primary bile acids but also elevated the levels of secondary bile acids. Notably, it enhanced the conversion of taurocholic acid (TCA) to deoxycholic acid (DCA), leading to a balanced bile acid metabolism. Finally, cecal contents of SJT-treated mice showed a significant reduction in the growth of C. difficile, underscoring the therapeutic potential of SJT via modulation of gut microbiota and bile acid metabolism. CONCLUSION: SJT demonstrates remarkable efficacy in treating CDI in mice by not only effectively combating the infection but also restoring the intricate balance of gut microbiota and bile acid metabolism. Furthermore, promising indications suggest that SJT may have the potential to prevent CDI recurrence. These findings underscore the comprehensive therapeutic value of SJT in managing CDI. Moving forward, we plan to transition from the laboratory to clinical settings to conduct further studies, validating our conclusions on SJT's efficacy.