Introduction and Objective: Since the 1980s, metronidazole (MTZ) has been used to treat C. difficile infection (CDI). Reduced clinical success of MTZ has been widely observed, but the reason for treatment failure remained unknown. Hence, we investigated MTZ resistance and the molecular basis of MTZ resistance. Materials and Methods: We screened for MTZ resistance using our improved susceptibility testing method. MTZ resistance via heme modulation was studied using RNA-seq. A transposon (Tn) mutant library of resistant strain was created and screened for loss of resistance phenotype. Genome-wide analysis was performed on >200 isolates, comprising both resistant and susceptible isolates. Results: Sixty-one percent of clinical isolates were MTZ-resistant, and modulated by heme. In a resistant epidemic strain, heme significantly dampened extensive stress-response gene perturbations caused by exposure to MTZ but had no effect in a susceptible historic strain. Screening of ~7000 Tn mutants led to the identification of an MTZ-susceptible mutant (MIC=0.5 µg/ml; WT MIC=4 µg/ml), with a unique mutation in 5-nitroimidazole reductase (nimB). Genome-wide SNP analysis revealed that resistance emerged through the development of this mutation. Evolutionary analysis showed that this mutation coevolved with gyrA mutation, associated with hypervirulent epidemic C. difficile. Conclusion and Significance/Implication: Unfortunately, MTZ, an affordable antibiotic, is no longer recommended for CDI therapy due to high treatment failure. This study provides the first direct evidence behind the elusive clinical failure of MTZ in CDI patients and the molecular factors driving the global epidemiology of the notorious hypervirulent MTZ-resistant C. difficile strains.