We identified a gene encoding the N-acetylmuramoyl L-alanine amidase (amiB) of Vibrio anguillarum, which catalyzes the degradation of peptidoglycan in bacteria. The entire open reading frame (ORF) of the amiB gene was composed of 1,722 nucleotides and 573 amino acids. The deduced amino acid sequence of AmiB showed a modular structure with two main domains; an N-terminal region exhibiting an Ami domain and three highly conserved, continuously repeating LysM domains in the C-terminal portion. An amiB mutant was constructed by homologous recombination to study the biochemical function of the AmiB protein in V. anguillarum. Transmission electron microscopy (TEM) revealed morphological differences, and that the mutant strain formed trimeric and tetrameric unseparated cells, suggesting that this enzyme is involved in the separation of daughter cells after cell division. Furthermore, inactivation of the amiB gene resulted in a marked increase of sensitivity to oxidative stress and organic acids.