The peptidoglycan is essential for structural maintenance and osmotic protection of the bacterial cell and the main target of numerous antibiotics. Amidst continuous remodeling, the old peptidoglycan mesh needs to be disconnected - particularly during cell division. Vibrio parahaemolyticus encodes several M23 endopeptidases, a class of enzymes involved in this process. We show that a previously uncharacterized protein displays D,D endopeptidase activity and is involved in cell separation. Upon deletion, cells exhibit a division phenotype, resulting in drastically decreased cell viability and increased Polymyxin sensitivity. Interestingly this phenotype is unique to a single M23 domain protein deletion in V. parahaemolyticus.