[Objective] To explore the role of lipopolysaccharide in Avian pathogenic Escherichia coli (APEC). [Methods] By using λ-phage Red recombination system, we generated the lipid A biosynthesis associated genes lpxL and lpxM mutants E516ΔlpxL, E516ΔlpxM, E516ΔlpxLΔlpxM, E522ΔlpxL, E522ΔlpxM and E522ΔlpxLΔlpxM. We conducted a series of in vivo and in vitro assays to investigate their biological characteristics and pathogenicity. [Results] The growth rate of the strains was basically the same. The ability of resistance to serum and killing by chicken macrophages were significantly impaired, E516ΔlpxL, E516ΔlpxLΔlpxM, E522ΔlpxM and E522ΔlpxLΔlpxM were significantly attenuated than those of the wild-type strains, and E516ΔlpxM and E522ΔlpxL had no obvious difference in relation to their parent strains. LD₅₀ results show that the pathogenicity of mutants was significantly attenuated than those of the wild-type strains. The colonization and survival model demonstrated that the loads of mutants were significantly decreased compared with those of the wild-type strains in all bloods and organs tested in chickens. The ability of mutants to enter and survive in chicken macrophage HD-11 is significantly reduced compared with those of the parent strains. [Conclusion] These results indicated that lpxL and lpxM genes are critical to the pathogenicity of APEC E516 and E522, lpxL had a more significant effect on virulence of APEC E516, and lpxM gene on virulence of APEC E522.