Bacterial lipopolysaccharide (LPS) molecules are an important cell surface component that enables adhesion to surfaces and cell motility, amongst other functions. InEscherichia coli, there are multiple Heptosyltransferase enzymes involved in the biosynthesis of the core region of LPS. Here we describe the first ever cloning, expression, purification and characterization of Heptosyltransferase III (HepIII) fromE. coli, which catalyzes the addition of an l-glycero-d-manno-heptose (Hep) residue to the growing LPS core via an α(1 → 7) bond. Inspired by results from our lab on theE. coliHepI, we assessed the catalytic efficiency with phospho-Hep2-Kdo2-Lipid A (PH2K2LA) and two deacylated analogues.