Peptidoglycan is a cell-sized polymer that surrounds the plasma membrane of a vast majority of bacterial cells, forming a semi-rigid cell wall that is required for maintaining cellular integrity and shape. It represents a major target for diverse antibacterial agents, including antibiotics, host-derived antimicrobial peptides, and bacteriocins, making it the “Achilles heel” of bacteria. Peptidoglycan synthesis relies on the translocation of its disaccharide-peptide building block across the plasma membrane. This process begins with the assembly of the peptidoglycan subunit onto a lipid carrier, typically undecaprenyl phosphateUndecaprenyl phosphate. The enzymes MraY and MurG sequentially catalyze the transfer of phosphoryl-N-acetylmuramoyl-pentapeptide and N-acetylglucosamine moieties onto the lipid carrier to form the membrane-bound intermediate lipid II. Before translocation, lipid II may undergo species-specific modifications, such as the addition of amino acids, peptides, or other functional groups to its peptide stem. The complete lipid II is then flipped to the outer leaflet of the membrane by the MurJ flippase. At the cell surface, a group of enzymes collectively known as peptidoglycan polymerases incorporate the subunit into nascent glycan strands and cross-link them with the existing mesh-like network, in coordination with peptidoglycan-degrading enzymes called autolysins, which constantly remodel the cell wall. The lipid carrier is produced via a two-step pathway: UppS catalyzes the elongation of the isoprenoid chain to yield undecaprenyl pyrophosphate, which is subsequently dephosphorylated by an as-yet unidentified enzyme. During peptidoglycan polymerization, the lipid carrier is released in its pyrophosphate form that must be recycled to sustain the high demand for cell wall precursors. This recycling is initiated by BacA and PAP2 family members, which act as integral membrane undecaprenyl pyrophosphate phosphatases. The resulting lipid is then flipped back to the cytoplasmic side by membrane-embedded proteins from the DedA family or DUF368-containing proteins, enabling a new cycle of synthesis. Collectively, the biosynthesis of membrane-bound peptidoglycan precursors, their incorporation into the growing cell wall, and the regeneration of the lipid carrier constitutes the lipid II cycle.