Members of the Rickettsia genus are obligate intracellular, Gram-negative coccobacilli that infect mammalian and arthropod hosts. Several rickettsial species are human pathogens and are transmitted by blood-feeding arthropods. In mouse infection models, control of rickettsial burden and disease resolution depends upon inflammatory cytokine production that is driven, in part, by Toll-like receptor (TLR) activation. The lipid A component of Gram-negative lipopolysaccharide (LPS) is the classical agonist of TLR4, however lipid A structure of rickettsial LPS and its inflammatory potential are unknown. Here we report the structure of lipid A from several species of Rickettsia including fatty acid analysis of Rickettsia rickettsii str. Sheila Smith, the most virulent species and the etiological agent of Rocky Mountain Spotted Fever. Furthermore, we have identified and characterized a new member of the recently discovered LpxJ family, a late-acyltransferase in Rickettsia typhi, the etiological agent of murine typhus, and R. rickettsii. Our results demonstrate that this enzyme, LpxJ, catalyzes the addition of a secondary acyl chain (C16) to the 3'-linked primary acyl chain of the lipid A moiety in the last step of the Raetz pathway of lipid A biosynthesis. Since lipid A architecture is fundamental to bacterial outer-membrane integrity, we believe LpxJ is important in maintaining ideal membrane dynamics to facilitate molecular interactions at the host-pathogen interface that are required for adhesion and invasion of mammalian cells. This work promises to reveal novel insights into Rickettsia pathogenesis and contribute greatly to our understanding of rickettsial physiology.