The O-antigen is one of the most variable Gram-negative cell constituents, and its specificity is important for bacterial niche adaptation. The observed diversity of O-antigen forms is mainly due to genetic variations in O-antigen gene clusters. Less common is a change of gene function due to nucleotide substitution; a new instance of which is reported here. The O-antigens of E. coli O107 and O117 have similar structures differing only in a single sugar residue (GlcNAc in O107 substituted for Glc in O117). These O-antigen gene clusters contain the same set of 11 genes and share 98.6% overall DNA identity. The function of the genes in the gene clusters have been proposed previously, and a glycosyltransferase gene (wclY) with nucleotide polymorphism in each strain was proposed to transfer different sugars in different strains. To identify the gene responsible for the transfer of different sugars, wclY mutants of E. coli O107 and O117 were constructed, and each mutant was complemented with the wclY genes cloned from both O107 and O117. Structural analysis of the O-antigens of the four recombinant strains identified wclY as a Glc-transferase in O117 and a GlcNAc-transferase in O107. The evolutionary relationship of E. coli O107 and O117 O-antigens is also discussed.