Clostridioides difficile, the leading cause of antibiotic-associated diarrhea, relies primarily on 3-3 crosslinks created by L,D-transpeptidases (LDTs) to fortify its peptidoglycan (PG) cell wall. This is unusual, as in most bacteria the vast majority of PG crosslinks are 4-3 crosslinks, which are created by penicillin-binding proteins (PBPs). Here we report the unprecedented observation that 3-3 crosslinking is essential for viability in C. difficile. We also report the discovery of a new family of LDTs that use a VanW domain to catalyze 3-3 crosslinking rather than a YkuD domain as in all previously known LDTs. Bioinformatic analyses indicate VanW domain LDTs are less common than YkuD domain LDTs and are largely restricted to Gram-positive bacteria. Our findings suggest that LDTs might be exploited as targets for antibiotics that kill C. difficile without disrupting the intestinal microbiota that is important for keeping C. difficile in check.