Clostridium difficile infection (CDI) has become the largest single cause of hospital-acquired infection in the United States. A compromised gut microbiota, typically through recent antibiotic exposure, is a prerequisite of C. difficile colonization susceptibility. This has been described for multiple antibiotic classes, which many result in distinct gut communities and each present individual metabolic challenges to C. difficile. We hypothesized that C. difficile must adapt its physiology to nutrient availability within the gut. Utilizing an in vivo model of CDI with three separate antibiotic pretretments, we demonstrated C. difficile highly colonized the cecum of mice in each group. Levels of spore and toxin production varied between treatments, both processes having been linked nutrient concentrations. To more closely investigate specific responses of C. difficile during infection, we performed targeted transcriptional analysis from cecal content of infected mice. This revealed variation in expression of life-cycle switches and catabolic pathways for various carbon sources. In order to assess which substrates C. difficile was exploiting, we further characterized the systems using transcriptomic-enabled genome-scale metabolic modeling and untargeted metabolomic analysis. Through development of a novel metabolite scoring algorithm, leveraging the metabolic model architecture, we were able to infer that a given metabolite was acquired from the environment. Our results support our hypothesis that C. difficile occupies alternative nutrient niches by metabolizing separate carbohydrate sources in each infection and these distinctions track with disparity in pathogenicity. Additionally, these data highlight conserved elements of C. difficile's metabolic strategy, including consumption of N-acetyl-D-glucosamine and Stickland fermentation substrates.