Clostridioides difficile(CD) is a sporulating and toxin-producing nosocomial pathogen that opportunistically infects the gut, particularly in patients with depleted microbiota after antibiotic exposure. Metabolically, CD rapidly generates energy and substrates for growth from Stickland fermentations of amino acids, with proline being a preferred reductive substrate. To investigate thein vivoeffects of reductive proline metabolism onC. difficile’svirulence in an enriched gut nutrient environment, we evaluated wild-type and isogenicΔprdBstrains of ATCC43255 on pathogen behaviors and host outcomes in highly susceptible gnotobiotic mice. Mice infected with theΔprdBmutant demonstrated extended survival via delayed colonization, growth and toxin production but ultimately succumbed to disease.In vivotranscriptomic analyses demonstrated how the absence of proline reductase activity more broadly disrupted the pathogen’s metabolism including failure to recruit oxidative Stickland pathways, ornithine transformations to alanine, and additional pathways generating growth-promoting substrates, contributing to delayed growth, sporulation, and toxin production. Our findings illustrate the central role for proline reductase metabolism to support early stages ofC. difficilecolonization and subsequent impact on the pathogen’s ability to rapidly expand and cause disease.