During bacterial cell division, amidases hydrolyse peptidoglycan to allow daughter cell separation. During the final stages of cell division, septal peptidoglycan between the two daughter cells prevents cell separation. Proteins called Nacetylmuramoyl- L-alanine type 3 amidases hydrolyse the amide bond between glycan strands. This allows successful daughter cell separation. This hydrolase activity is tightly regulated by a pathway of protein interactions to avoid uncontrolled peptidoglycan degradation. The exact structural mechanism of the protein interaction pathway involved in regulating the activity of such amidases is currently unknown. Here it is shown, the molecular regulatory mechanism by which, Escherichia coli amidase, AmiA, is activated by the interaction of EnvC. It was found that EnvC, an amidase regulator, is self-regulated by a restraining helix over the amidase binding LytM domain evidenced by bacterial-two-hybrid and co-purification analysis of EnvC truncated variants. By solving a 2.35 angstrom structure of E. coli and building an AmiA bound to EnvC LytM domain model, the molecular mechanism of the removal of a blocking helix over AmiA’s active site by the interaction of an interaction helix in AmiA to EnvC. These results show a stringently structurally regulated amidase activation pathway, with no availability of the amidase active site without interaction with a regulator protein. This is a potentially conserved regulatory system that could be use to model amidase regulation in other bacterial species. As a part of fundamental biology in a model bacterial system, it is anticipated that the understanding found in this research could extend to understanding related cell division pathways in other bacterial species. As an application of this regulation mechanism, there is a potential antibiotic target to be found in cell division amidases, as without self-regulation, amidases may uncontrollably hydrolyse peptidoglycan, weakening the integrity of cells to make them more susceptible to other antibiotic treatments.