Vibrio cholerae O1 employs the ATP-binding cassette (ABC) transporter-dependent pathway for O antigen biosynthesis. Different from highly studied Klebsiella pneumoniae and Escherichia coli, it was reported that initial reaction of O antigen biosynthesis in V. cholerae O1 may be involved in WbeW protein, which is predicted to be a galactosyltransferase. In this work, we report expression and characterization of WbeW enzyme. WbeW was expressed as membrane-associated form in E. coli and it was obtained with high purity. The enzyme had a function of transferring Gal-1-P from UDP-Gal to Und-P, implying that initial glycan of O antigen in V. cholerae O1 can be composed of a Gal residue.