The lipopolysaccharide (LPS) macromolecule is the major constituent of the external leaflet of the Gram-negative outer membrane, exerting a plethora of biological activities in animals and plants. Among all, it represents a defensive barrier which helps bacteria to resist antimicrobial compounds and external stress factors and is involved in most aspects of host–bacterium interactions such as recognition, adhesion and colonization. One of the most interesting and studied LPS features is its key role in the pathogenesis of Gram-negative infections potentially causing fever or circulatory shock. On the other hand, the LPS acts as a beneficial factor for the host since it is recognized by specific receptors of the host innate immune system; this recognition activates the host defenses culminating, in most cases, in destruction of the pathogen. Most of the biological roles of the LPS are strictly related to its primary structure; thus knowledge of the structural architecture of such a macromolecule, which is different even among bacterial strains belonging to the same species, is a first step but is essential in order to understand the molecular bases of the wide variety of biological activities exerted by LPSs.