Shigella strains are human pathogens and normally identified based on their O antigens. The chemical structure and gene cluster of Shigella boydii type 17 O antigen were studied. As judged by sugar and methylation analyses along with NMR spectroscopy data, the O antigen of S. boydii type 17 has a linear trisaccharide O unit, which consists of two residues of N-acetylgalactosamine (GalNAc) and a 4-O-[(R)-1-carboxyethyl]-d-glucose (glucolactilic acid). The O antigen gene cluster of S. boydii type 17 was sequenced and genes encoding UDP-N-acetylglucosamine C4 epimerase for GalNAc synthesis, O unit flippase, O antigen polymerase, and glycosyltransferases were putatively identified based on sequence similarities and the presence of conserved motifs. Two genes, whose functions could not be clearly indicated by homology search, were confirmed to be involved in the synthesis of glucolactilic acid by mutation and structural verification of the O antigens from the mutants. To our knowledge, this is the first time that genes involved in the synthesis of glucolactilic acid have been reported. Two genes specific to S. boydii type 17 were also identified.