An intact gut microbiota confers colonization resistance against Clostridioides difficile through a variety of mechanisms, likely including competition for nutrients. Recently, proline was identified as an important environmental amino acid that C. difficile uses to support growth and cause significant disease. A posttranslationally modified form, trans-4-hydroxyproline, is highly abundant in collagen, which is degraded by host proteases in response to C. difficile toxin activity. The ability to dehydrate trans-4-hydroxyproline via the HypD glycyl radical enzyme is widespread among gut microbiota, including C. difficile and members of the commensal Clostridia, suggesting that this amino acid is an important nutrient in the host environment. Therefore, we constructed a C. difficile ΔhypD mutant and found that it was modestly impaired in fitness in a mouse model of infection, and was associated with an altered microbiota when compared to mice challenged with the wild-type strain. Changes in the microbiota between the two groups were largely driven by members of the Lachnospiraceae family and the Clostridium genus. We found that C. difficile and type strains of three commensal Clostridia had significant alterations to their metabolic gene expression in the presence of trans-4-hydroxyproline in vitro. The proline reductase (prd) genes were elevated in C. difficile, consistent with the hypothesis that trans-4-hydroxyproline is used by C. difficile to supply proline for energy metabolism. Similar transcripts were also elevated in some commensal Clostridia tested, although each strain responded differently. This suggests that the uptake and utilization of other nutrients by the commensal Clostridia may be affected by trans-4-hydroxyproline metabolism, highlighting how a common nutrient may be a signal to each organism to adapt to a unique niche. Further elucidation of the differences between them in the presence of hydroxyproline and other key nutrients will be important in determining their role in nutrient competition against C. difficile. IMPORTANCE Proline is an essential environmental amino acid that C. difficile uses to support growth and cause significant disease. A posttranslationally modified form, hydroxyproline, is highly abundant in collagen, which is degraded by host proteases in response to C. difficile toxin activity. The ability to dehydrate hydroxyproline via the HypD glycyl radical enzyme is widespread among gut microbiota, including C. difficile and members of the commensal Clostridia, suggesting that this amino acid is an important nutrient in the host environment. We found that C. difficile and three commensal Clostridia strains had significant, but different, alterations to their metabolic gene expression in the presence of hydroxyproline in vitro. This suggests that the uptake and utilization of other nutrients by the commensal Clostridia may be affected by hydroxyproline metabolism, highlighting how a common nutrient may be a signal to each organism to adapt to a unique niche. Further elucidation of the differences between them in the presence of hydroxyproline and other key nutrients will be important to determining their role in nutrient competition against C. difficile.