Protozoan grazing is a major cause of bacterial mortality and controls bacterial population size and composition in the natural environment. To enhance their survival, bacteria evolved many defense strategies to avoid grazing by protists. Cell wall modification is one of the defense strategies that helps bacteria escape from recognition and/or internalization by its predators. Lipopolysaccharide (LPS) is the major component of Gram-negative bacterial cell wall. LPS is divided into three regions: lipid A, oligosaccharide core and O-specific polysaccharide. O-polysaccharide as the outermost region ofE. coliLPS provides protection against predation byAcanthamoeba castellanii; however, the characteristics of O-polysaccharide contribute to this protection remain unknown. Here, we investigate how length, structure and composition of LPS affectE. colirecognition and internalization byA. castellanii. We found that length of O-antigen does not play a significant role in regulating bacterial recognition byA. castellanii. However, the composition and structure of O-polysaccharide play important roles in providing resistance toA. castellaniipredation.