Abstract Clostridium difficile infection (CDI) is the most prevalent and devastating healthcare-associated diarrheal infection, with a 1%-2.5% mortality rate, translating to 15,000-30,000 deaths annually in the US. Repeated episodes of CDI are common and are difficult to treat, causing a significant hardship for patients. Remarkably, fecal microbiota transplant (FMT), a measure in which the entire fecal microbiota from a healthy donor is transferred to an infected individual, cures more than 90% of individuals with recurrent CDI. FMT?s high efficacy provides a powerful insight into the importance of gut microbiota in resistance against C. difficile, and offers the promise that microbial factors interfering with C. difficile could be identified and exploited for microbiota-based therapies. However, manipulating and engineering microbiota as a preventive or therapeutic strategy is still beyond our reach, due, in part, to the uncertainty of the identity and functions of specific microbes that confer resistance against C. difficile. We hypothesize that select members of cultivable murine and human-associated Lachnospiraceae and Ruminococcaceae are sufficient to confer C. difficile resistance in a gnotobiotic C. difficile challenge mouse model. This hypothesis is supported by recent literature and our own preliminary data that: (1) Lachnospiraceae and Ruminococcaceae, two major families of the Clostridia class within the Firmicutes phylum in the indigenous microbiota, are depleted in the gut microbiome of C. difficile patients, suggesting that these two bacterial families harbor protective functions against C. difficile; (2) using germ-free (GF) mice colonized with serial dilutions of mouse microbiota, we discovered that eliminating ~15 dominant members of Lachnospiraceae renders mice susceptible to C. difficile. Together, these results provide compelling evidence that we could leverage GF mice gavaged with cultivable members of murine or human microbiota at varying degrees of richness to isolate cultivable commensals with C. difficile resistance phenotype. Our Specific Aims are: 1) Identify cultivable members of murine Lachnospiraceae and Ruminococcaceae conferring resistance to C. difficile, and 2) Identify cultivable members of human Lachnospiraceae and Ruminococcaceae with C. difficile resistance phenotype. Our approach is innovative because it will utilize culturomics and gnotobiotic mice to target specific murine and human microbes that confer resistance against C. difficile. The proposed research is significant because there is an urgent and critical need to isolate members of protective indigenous microbiota for mechanistic studies of C. difficile resistance and human application. Our combined approach will cultivate murine and human-associated microbes that can be directly tested in mice for mechanistic studies, and translated to humans to enable clinical application, ultimately facilitating the development for novel microbiota-based therapeutics for CDI.