SignificancePeptidoglycan amidases break the peptidoglycan layer during cell division and maintain integrity of the cell envelope. Here, we present structures of an isolated peptidoglycan hydrolase in an autoinhibited (“off”) state and a second amidase bound to the activating LytM domain of EnvC revealing the active (“on”) state. A comparison of these structures provides important molecular insights into the activation of peptidoglycan hydrolases by their cognate activators.