Clostridioides difficile is the leading cause of nosocomial infections in the United States and costs the healthcare system an estimated $5 billion/yr. C. difficile infections are costly and difficult to treat because they recur at high frequency (~20%). Disease recurrence depends on C. difficile’s ability to form metabolically dormant spores because they are the transmissive form of this obligate anaerobe. Recent studies in mice have shown that preventing spore formation can break the damaging cycle of recurrent infection that characterizes C. difficile disease. While blocking spore formation with cephamycins can prevent recurrence in mice when combined with vancomycin, the current standard-of-care, cephamycins can sensitize humans to C. difficile infections by exacerbating gut dysbiosis. Thus, anti-sporulation therapies that selectively target C. difficile are needed. Developing such therapies, however, will require a deeper understanding of how C. difficile assembles a spore. Cephamycins block spore formation by inhibiting SpoVD, a sporulation-induced penicillin-binding protein. In Bacillus subtilis, SpoVD works in concert with the SpoVE glycosyltransferase and SpoVB flippase to synthesize a thick, protective layer of spore peptidoglycan (PG) known as the cortex. While we confirmed that C. difficile cortex synthesis requires these three factors, we unexpectedly found that C. difficile SpoVD and SpoVE regulate the earliest stage of spore formation, asymmetric division. We also identified sporulation-induced, divisome-like proteins that regulate asymmetric division. These results strongly suggest that C. difficile uses a unique sporulation-induced PG synthesis machine to synthesize polar septa; this machinery could be selectively targeted to prevent spore formation. Our data further suggest that C. difficile uses a distinct PG synthesis machinery to synthesize medial septa during vegetative cell division because the divisome components we have identified are dispensable in C. difficile, despite being essential in all other bacteria studied to date. Interestingly, sporulation-induced SpoVD may modulate vegetative cell division because loss of SpoVD sensitizes C. difficile to cephamycin antibiotics during broth culture. Based on these findings, this proposal seeks to determine how C. difficile regulates polar septum formation during sporulation and how it uses some of these components to enhance C. difficile’s resistance to cell wall antibiotics during vegetative growth. Completing these aims will define an important new mechanism by which C. difficile assembles infectious spores, lay the foundation for developing C. difficile-specific anti-sporulation therapies, and reveal novel mechanisms that contribute to C. difficile’s high level of resistance to cell wall antibiotics.