Lipopolysaccharide is the main component of outer membranes, which serve as a permeability barrier. In this study, we investigated the role of the structure of lipopolysaccharide on the permeability of bacterial cell membranes. Nine Escherichia coli strains which make different structures of LPS were selected or constructed. Lipopolysaccharide and lipid A were extracted from these strains, and their structures were analyzed by using thin-layer chromatography and electrospray ionization mass spectrometry. The membrane permeability of these strains was analyzed by using N-phenyl-1-naphthylamine fluorescent probe. Wild type E. coli showed the least permeability, while mutants in which LPS structures were changed by the deletion or expression of related genes showed higher permeability. The number of phosphate groups and the acyl chains, and the length of the polysaccharide of lipopolysaccharide all affect the permeability of E. coli. The chain length of the polysaccharide has the largest effect on the permeability, followed by the number of the acyl chains. The results indicate that the cellular membrane permeability is related to the structure of lipopolysaccharide.