The subject of this chapter is d-ala-d-ala Carboxypeptidase VanY. The VanY carboxypeptidase releases the C-terminal hydrolyses the d-ala from the precursor peptides that provide the crosslinks in the bacterial cell wall peptidoglycans. The enzyme has been characterized from a strain of the pathogenic bacterium Enterococcus faecium that is insensitive to the antibiotic vancomycin. Vancomycin binds to the d-Ala-d-Ala portion of the crosslinking peptide, preventing a transpeptidase from making the crosslinks in the peptidoglycan. The vancomycin resistance genes are encoded on a plasmid, and the VanH and VanA proteins produce the depsipeptide d-ala-d-lactate instead of d-ala-d-Ala. VanX degrades d-ala-d-Ala, pushing the equilibrium toward d-ala-d-lactate. d-ala-d-lactate can be used to crosslink the peptidoglycan but has a lower affinity for vancomycin, hence the resistance to the antibiotic. Unlike VanX, VanY is a penicillin-binding protein and forms a stable complex with benzylpenicillin. VanY increases tolerance to vancomycin but is dispensable in the presence of the vanHAX genes.