Previous study showed that some Gram-negative bacteria possess human blood group activity. Among them, Escherichia coli O86 has high blood group B activity and weak blood group A activity. This is due to the cell surface O-antigen structure, which resembles that of human blood group B antigen. In this study, we sequenced the entire E. coli O86 antigen gene cluster and identified all the genes responsible for O-antigen biosynthesis by sequence comparative analysis. The blood group B-like antigen in E. coli O86 O-polysaccharide was synthesized by sequentially employing three glycosyltransferases identified in the gene cluster. More importantly, we identified a new bacterial glycosyltransferase (WbnI) equivalent to human blood group transferase B (GTB). The enzyme substrate specificity and stepwise enzymatic synthesis of blood group B-like antigen revealed that the biosynthetic pathway of B antigen is essentially the same in E. coli O86 as in humans. This new finding provides a model to study the specificity and structure relationship of blood group transferases and supports the hypothesis of anti-blood group antibody production by bacterial stimulation.