Abstract: O-Antigens is the outermost component of bacterial lipopolysaccharide (LPS). Their structural complexity and diversity are essential for bacterial immune evasion, pathogenicity, and bacteria-host interactions. In this study, the O-antigen of lipopolysaccharide from Escherichia coli strain EPEC080 was extracted and purified. The chemical structure of the O-antigen was determined by a combination of various analytical methods, including molecular weight determination, monosaccharide composition analysis, infrared spectrum and nuclear magnetic resonance analysis. The O-antigen with an average molecular weight of 14.3 kDa was determined to consist of a pentasaccharide repeating unit: →4)-α-D-GalpNAc-(1→3)-α-L-Rhap-(1→2)-β-D-GalpNAc-(1→3)-α-L-Rhap-(1→2)-α-L-Rhap-(1→. Notably, this structure is different from those of known E. coli LPS O-antigens, indicating that strain EPEC080 represents a novel E. coli serotype. The structural identification of this O-antigen provides a foundation for further investigation of the pathogenesis of enteropathogenic E. coli and development of prophylaxis and treatment methods against the related diseases.