•MS provides valuable information about in vitro characterization of glycosyltransferases.•MS is highly valuable for precise structure elucidation of lipooligosaccharides.•The wbuP gene of E. coli O114 encodes a new β-1,3-Gal-transferase.•Nonionic surfactants exert a strong inhibitory effect on the activity of WbuP.In this study, synthetic acceptor substrate GlcNAc alpha-PO3-PO3-(CH2)11-O-phenyl (GlcNAc-PP-PhU) was employed in glycosyl transferase assays to characterize the WbuP galactosyltransferase activity. This activity was time- and enzyme concentration-dependent. The optimal enzyme activity was observed at pH 6.5 and 25 °C. The enzyme requires Mn2+ ions for maximal activity and detergents in the assay did not increase glycosyltransfer activity. The enzyme was shown to be specific for the UDP-Gal donor substrate. Kinetic parameters were determined for UDP-Gal, and GlcNAc-PP-PhU. The enzyme product was determined to have a β-1,3-linkage using strategies based on exoglycosidase digestion combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) as well as collision-induced dissociation electrospray ionization ion trap multiple tandem MS (CID-ESI-IT-MSn). Our results conclusively demonstrate that the wbuP gene of Escherichia coli O114 encodes a UDP-Gal: GlcNAc α-pyrophosphate-lipid β-1,3-Gal-transferase that transfers the second sugar moiety in the assembly of the O114 repeating unit.