ABSTRACT O antigen (O polysaccharide) is an important and highly variable cell component present on the surface of cells which defines the serospecificity of Gram-negative bacteria. Most O antigens of Shigella flexneri , a cause of shigellosis, share a backbone composed of →2)-α- l -Rha p III -(1→2)-α- l -Rha p II -(1→3)-α- l -Rha p I -(1→3)-β- d -Glc p NAc-(1→ repeats, which can be modified by adding various substituents, giving rise to 19 serotypes. The known modifications include glucosylation on various sugar residues, O-acetylation on Rha I , and phosphorylation with phosphoethanolamine on Rha II or/and Rha III . Recently, two new O-antigen modifications, namely, O-acetylation at position 3 or 4 of Rha III and position 6 of GlcNAc, have been identified in several S. flexneri serotypes. In this work, the genetic basis for the 3/4-O-acetylation on Rha III was elucidated. Bioinformatic analysis of the genome of S. flexneri serotype 2a strain Sf301, which carries 3/4-O-acetylation on Rha III , revealed an O-acyltransferase gene designated oacB . Genetic studies combined with O-antigen structure analysis demonstrated that this gene is responsible for the 3/4-O-acetylation in serotypes 1a, 1b, 2a, 5a, and Y but not serotype 6, which has a different O-antigen backbone structure. The oacB gene is carried by a transposon-like structure located in the proA-adrA region on the chromosome, which represents a novel mechanism of mobilization of O-antigen modification factors in S. flexneri . These findings enhance our knowledge of S. flexneri O-antigen modifications and shed light on the origin of new O-antigen variants.