•The binding of anti-Vi mAb and polyclonal immune sera correlated with the level of O-acetylation.•C. freundii Vi resists de-O-acetylation and is more viscous than S. Typhi Vi.•Sera from human vaccine recipients contains IgG that recognizes the backbone of Vi.•Simulations show O-acetyls are exposed on the surface of Vi and confer rigidity.•MD gives conformational rationale for effect of O-acetylation on Vi antigenicity and viscosity. In this work, we explore the effects of O-acetylation on the physical and immunological characteristics of the WHO International Standards of Vi polysaccharide (Vi) from both Citrobacter freundii and Salmonella enterica serovar Typhi. We find that, although structurally identical according to NMR, the two Vi standards have differences with respect to susceptibility to de-O-acetylation and viscosity in water. Vi standards from both species have equivalent mass and O-acetylation-dependent binding to a mouse monoclonal antibody and to anti-Vi polyclonal antisera, including the WHO International Standard for human anti-typhoid capsular Vi PS IgG. This study also confirms that human anti-Vi sera binds to completely de-O-acetylated Vi. Molecular dynamics simulations provide conformational rationales for the known effect of de-O-acetylation both on the viscosity and antigenicity of the Vi, demonstrating that de-O-acetylation has a very marked effect on the conformation and dynamic behavior of the Vi, changing the capsular polysaccharide from a rigid helix into a more flexible coil, as well as enhancing the strong interaction of the polysaccharide with sodium ions. Partial de-O-acetylation of Vi revealed hidden epitopes that were recognized by human and sheep anti-Vi PS immune sera. These findings have significance for the manufacture and evaluation of Vi vaccines.