Spores are produced by many organisms as the result of a survival mechanism, triggered under several types of adverse environmental conditions. They are multi-layered structures, composed of a compressed dehydrated inner core, surrounded by the inner membrane, a germ cell-wall, a peptidoglycan layer known as the cortex, an outer membrane, a proteinaceous external coat, and for some species the outermost layer called the exosporium. This study focuses on the spore cortex of Clostridium difficile, a Gram-positive spore-forming, toxin-producing anaerobic bacterium that can colonize the intestinal tracts of humans, considered as the leading cause of hospital and community-acquired antibiotic-associated diarrhea. Given the highly original structure described for the vegetative cell peptidoglycan of C. difficile, and notably the particularly high level of N-deacetylation and its impact on host-pathogen interactions, we focused on the cortex N-deacetylases, and especially the N-deacetylase responsible for muramic lactam synthesis in C. difficile. Moreover, given the central role of spores in the physiopathology of C. difficile infection, we also investigated the contribution of cortex structure in C. difficile virulence, presenting the first study connecting cortex structure and virulence. In this context, we provide the fine structure of C. difficile cortex and the characterization of pdaA as the N-deacetylase responsible for muramic lactams synthesis.