Abstract: Clostridioides difficile is the most prevalent cause of hospital associated infections (HAIs). As an obligately anaerobic pathogen, C. difficile can consume diverse carbon sources to support gut colonization and infection. In particular, glycolytic and Stickland amino acid fermentation pathways have been shown to support rapid growth, a finding that also occurs after antibiotic treatment elevates availability of these nutrient sources by removing competitive commensal species. However, the metabolic pathways of obligately anaerobic bacteria remain ill-defined, limiting capacity to identify preventive and therapeutic interventions that target C. difficile’s core physiology. To address these limitations, we will employ carbon-13 (13C) NMR of C. difficile’s dynamic metabolism to inform a genome-scale metabolic model and clearly define metabolic integration points among energy-generating glycolytic and amino acid fermentation pathways. Findings will be evaluated in targeted genetic mutants and with combinations of fermentable carbon sources to evaluate their impact on pathogen growth and virulence.