27In people, colonization with Clostridium difficile, the leading cause of antibiotic-associated diarrhea, has been shown to be 28 associated with distinct gut microbial features, including reduced bacterial community diversity and depletion of key taxa. In dogs, 29 the gut microbiome features that define C. difficile colonization are less well understood. We sought to define the gut microbiome 30 features associated with C. difficile colonization in puppies, a population where the prevalence of C. difficile has been shown to be 31 elevated, and to define the effect of puppy age and litter upon these features and C. difficile risk. We collected fecal samples from 32 weaned (n=27) and unweaned (n=74) puppies from 13 litters and analyzed the effects of colonization status, age and litter on 33 microbial diversity using linear mixed effects models. 34 Colonization with C. difficile was significantly associated with younger age, and colonized puppies had significantly decreased 35 bacterial community diversity and differentially abundant taxa compared to non-colonized puppies, even when adjusting for age. C. 36 difficile colonization remained associated with decreased bacterial community diversity, but the association did not reach statistical 37 significance in a mixed effects model incorporating litter as a random effect. 38 Even though litter explained a greater proportion (67%) of the variability in microbial diversity than colonization status, we 39 nevertheless observed heterogeneity in gut microbial community diversity and colonization status within more than half of the 40 litters, suggesting that the gut microbiome contributes to colonization resistance against C. difficile. The colonization of puppies with 3 41 C. difficile has important implications for the potential zoonotic transfer of this organism to people. The identified associations point 42 to mechanisms by which C. difficile colonization may be reduced. 43 44 45 46