Shigella flexneri is the most common cause of shigellosis in developing countries. Up to now, 23 serotypes of S. flexneri have been reported. Different serotypes result from the addition of acetyl, glucosyl, or phosphatidylethanolamine groups on the O-antigen backbone and horizontal transfer of mentioned groups can lead to serotype conversion among S. flexneri strains. Serotype conversion causes either a circulation of pre-existing serotypes or is responsible for the emergence of new serotypes. Serotype conversion plays a pivotal role in the protection and evasion of S. flexneri from the host immune response. Furthermore, spreading any new serotype can provide evolutionary advantages. Hence, information about S. flexneri O-antigen structure, serotype conversion, and serotyping methods can be helpful to understand the disease that attributes distinct serotypes in order to apply control or prevention methods in accordance with predominant serotypes over the course of time. Thus, the scope of this review is to give an overview of the serotype structures, factors involved in O-antigen modification, molecular analysis, and epidemiological evidence for the benefits of serotype conversion for S. flexneri serotypes. We are also providing a review of the typing methods.