The O antigen, consisting of many repeats of an oligosaccharide unit, is part of the lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria. It is on the cell surface and appears to be a major target for both immune system and bacteriophages, and therefore becomes one of the most variable cell constituents. The variability of the O antigen provides the major basis for serotyping schemes of Gram-negative bacteria. The genes responsible for the synthesis of O antigen are usually in a single cluster known as O antigen gene cluster, and their location on the chromosome within a species is generally conserved. Three O antigen biosynthesis pathways including Wzx/Wzy, ABC-transporter and Synthase have been discovered. In this chapter, the traditional and molecular O serotyping schemes are compared, O antigen structures and gene clusters of well-studied species are described, processes for formation and distribution of the variety of O antigens are discussed, and finally, the role of O antigen in bacterial virulence.