The outer membrane (OM) of Gram-negative bacteria provides an efficient barrier against external noxious compounds such as antimicrobial agents. Associated with drug target modification, it contributes to the overall failure of chemotherapy. In the complex OM architecture, Lipid A plays an essential role by anchoring the lipopolysaccharide in the membrane and ensuring the spatial organization between lipids, proteins, and sugars. Currently, the targets of almost all antibiotics are intracellularly located and require translocation across membranes. We report herein an integrated view of Lipid A synthesis, membrane assembly, a structure comparison at the molecular structure level of numerous Gram-negative bacterial species, as well as its recent use as a target for original antibacterial molecules. This review paves the way for a new vision of a key membrane component that acts during bacterial adaptation to environmental stresses and for the development of new weapons against microbial resistance to usual antibiotics.