Peptidoglycan monomers - consisting of the sugars NAM and NAG with a pentapeptide coming off the NAM - are synthesized in the cytosol of the bacterium. These monomers are then transported across the cytoplasmic membrane and inserted into the growing peptidoglycan chain by membrane transporters called bactoprenols.

Bacitracin binds to bactoprenol after it inserts the peptidoglycan monomer it is transporting into the growing cell wall. It subsequently prevents the dephosphorylation of the bactoprenol. Bactoprenol molecules that have not lost the second phosphate group cannot assemble new monomers and transport them across the cytoplasmic membrane. As a result, no new monomers are inserted into the growing cell wall. As the autolysins continue to break the peptide cross-links and new cross-links fail to form, the bacterium bursts from osmotic lysis.