Polysaccharides are essential and immunologically relevant components of bacterial cell walls. These biomolecules can be found covalently attached to lipids (e.g., O-polysaccharide (PS) contains undecaprenyl and lipopolysaccharide (LPS) contains lipid A) or noncovalently associated with cell wells (e.g., capsular PS (CPS)). Although extensive genetic studies have indicated that the Wzy-dependent biosynthetic pathway is primarily responsible for producing such polysaccharides, in vitro biochemical studies are needed to determine, for example, which gene product is responsible for catalyzing each step in the pathway, and to reveal molecular details about the Wzx translocase, Wzy polymerase and O-PS chain-length determinant. Many of these biochemical studies require access to a structurally well-defined PS repeating unit undecaprenyl pyrophosphate (RU-PP-Und), the key building block in this pathway. We describe herein the chemoenzymatic synthesis of Escherichia coli (serotype O157) RU-PP-Und. This involves (i) chemical synthesis of precursor N-acetyl-D-galactosamine (GalNAc)-PP-Und (2 weeks) and (ii) enzymatic extension of the precursor to produce RU-PP-Und (2 weeks). Undecaprenyl phosphate and peracetylated GalNAc-1-phosphate are prepared from commercially available undecaprenol and peracetylated GalNAc. The chemical coupling of these two products, followed by structural confirmation (mass spectrometry and NMR) and deprotection, generates GalNAc-PP-Und. This compound is then sequentially modified by enzymes in the E. coli serotype O157 (E. coli O157) O-PS biosynthetic pathway. Three glycosyltransferases (GTs) are involved (WbdN, WbdO and WbdP) and they transfer glucose (Glc), L-fucose (L-Fuc) and N-acetylperosamine (PerNAc) onto GalNAc-PP-Und to form the intact RU-PP-Und in a stepwise manner. Final compounds and intermediates are confirmed by mass spectrometry. The procedure can be adapted to the synthesis of analogs with different PS or lipid moieties.