The outer membrane of Gram-negative bacteria is an asymmetric bilayer in which lipopolysaccharide (LPS), the principal component of the outer leaflet, promotes tight packing and ordering of the membrane components that are essential for the barrier and load-bearing functions of this membrane. Lipopolysaccharide mobility is known to be restricted in the outer membrane, but this confinement and the underlying biophysical interactions responsible remain to be fully characterized. Here, we apply a bio-orthogonal strategy for in situ site-specific fluorescent labeling of LPS. Using fluorescence microscopy, we quantify LPS lateral confinement in the outer membrane of Escherichia coli and demonstrate that this confinement is independent of oligosaccharide domain structure. We show that lipopolysaccharide assembles into discrete supramolecular structures, and that restricted lateral mobility arises from a combination of divalent cation-mediated electrostatic interactions in the anionic Lipid A headgroup, and hydrophobic interactions between acyl chains within the lipid milieu. Magnesium cations exert a greater influence than calcium cations on lipopolysaccharide lateral mobility. These traits are conserved across multiple pathogenic bacterial species, irrespective of O-antigen serotype, showing that lipopolysaccharide confinement is a ubiquitous feature of Gram-negative bacteria.