O-acetylation of the essential bacterial heteropolymer peptidoglycan is required for modulation of host-pathogen interactions and proper cell growth. Given this, the O- acetylation pathway is thought to present a novel drug target. Our current understanding of the enzymes involved in this process lacks the key structural information required for better identification and design of novel inhibitors. This work demonstrates the production and purification of a stable and active recombinant form of peptidoglycan O-acetyltransferase B (PatB1) from Bacillus cereus for use in crystallization studies. The enzymatic function of PatB1 was demonstrated to be dependent on the presence of the sugar-based acetyl acceptor chitobiose, in a manner similar to PatB from Neisseria gonorrhoeae. Using a method which combines surface entropy reduction and in situ proteolysis, single crystals of PatB1 were produced in sparse matrix crystallization screens and diffracted to 2.0 Å resolution.