Antibiotic resistance is on the rise, making bacterial infections an increasing threat to human health. O-antigenic polysaccharides are important virulence factors of pathogenic Gram-negative bacteria that can be involved in immune evasion and colonization. The O antigens of enteropathogenic Salmonella enterica O43 (SO43) and Escherichia coli O86 (ECO86) are structurally similar and contain a mimic of the blood group B determinant. However, the SO43 O antigen repeating unit has GlcNAc at the reducing end while ECO86 contains a GalNAc residue. To explore this difference we characterized the α1,3-GalNAc-transferase responsible for the addition of GalNAc to GalNAc-PP-undecaprenol in ECO86 (WbnH) and the enzyme proposed to add GalNAc to GlcNAc-PP-undecaprenol in SO43 (WfbG). Substrate specificity study of these GT4 enzymes showed a strict donor specificity for UDP-GalNAc. However, WfbG could use either GlcNAcα- or GalNAcα-PP-phenylundecyl as a natural acceptor substrate analog whereas WbnH was only active with GalNAcα-PP-phenylundecyl. The GlcNAc-PP-undecaprenol 4-epimerase gene in the ECO86 strain can provide the essential acceptor substrate for WbnH. These data help to explain the difference in O antigen structures between SO43 and ECO86. A series of GT4 enzymes was analyzed by bioinformatics to identify common sequences that help to predict their functions. Characterization of these bacterial GTs can identify potential targets to disrupt virulence mechanisms.