Polysaccharides constitute a major component of the bacterial cell surface. They play critical roles in the interactions between bacteria and the host environments, and consequently contribute to the virulence of pathogens. The lipopolysaccharide (LPS) found on the surface of gram-negative bacteria consists of three parts: lipid A, a core oligosaccharide, and the O antigen. The O antigen is the outermost part of LPS and contains multiple oligosaccharide repeating units. Biosynthesis of the O-repeating unit is the first committed step in LPS biosynthesis. We sequenced and characterized the O-antigen biosynthetic gene cluster of Escherichia coli serotype O86. Four glycosyltransferases encoded by the genes within the cluster were cloned and overexpressed. In vitro reconstitution of the O-repeating unit of E. coli 086 was achieved via using these enzymes.