The O antigen side chain portion of lipopolysaccharide acts as a physical barrier to protect the surface of Gram-negative bacteria from components of the innate immune system. Genomes of the pathogen Salmonella enterica can contain up to four different glycosyltransferase (gtr) operons. The enzymes encoded by these gtr operons modify the O antigen subunit, typically by the addition of a glucose residue. These gtr operons often phase vary in an epigenetic manner, resulting in a mixed population with different O antigen structures on the surface. The human-adapted, invasive serovar Salmonella Typhi contains two such gtr operons, referred to as the Family 2 and Family 3 operons. Using strains constitutively expressing the Family 2 and Family 3 gtr operons, NMR analysis established that the Family 3 operon performs a previously characterized glucosylation of the galactose sugar in the basal subunit. The Family 2 operon performs an acetylation of the rhamnose residue; this is the first time this enzymatic activity has been associated with a gtr operon. Further investigation into the expression of these gtr operons in S. Typhi revealed a mutation in the promoter region to the Family 3 operon resulting in constitutive expression while the Family 2 operon maintained phase variable expression. The role these modifications play during an infection or how they influence the interaction with the host’s immune system has not been well studied, especially in regards to the invasive infection caused by S. Typhi. Using serum sensitivity assays, we determined that the presence of the glucosylation modification significantly increased survival within normal human serum while the acetylation modification had no effect. This survival advantage may explain why the S. Typhi Family 3 operon has evolved to have constitutive expression compared to the Family 3 gtr operons present in other serovars.