The Gram-negative bacterium Pasteurella multocida is the causative agent of many animal diseases, including fowl cholera. All P. multocida strains examined to date produce an O-antigen-deficient lipopolysaccharide (LPS). Our long term genetic and structural study on P. multocida LPS has revealed that the molecule can be decorated with phosphoethanolamine (PEtn) at a number of positions. These include the addition of PEtn to lipid A, 3-deoxy-D-manno-octulosonic acid (Kdo) or an inner core heptose. Unusually, PEtn can also be present on galactose residues at the distal end of the LPS molecule. Bioinformatic analysis of the highly virulent strains VP161 and X73 and their corresponding PEtn mutants, has allowed us to identify the full cohort of LPS serotype/genotype 1 PEtn transferase genes. PetL (required for addition of PEtn to lipid A) and Lpt_3 (required for the addition of PEtn to the second heptose) share a high degree of identity with PEtn transferases identified and characterized in other bacterial species. However, the galactose-specific PEtn transferase, PetG, and the PEtn transferase that transfers PEtn to the Kdo residue, PetP, have not been identified or characterized in any other species. Interestingly, all four PEtn genes are intact in X73 but lpt_3 and petG are present as pseudogenes in many other P. multocida strains (including the highly virulent VP161) indicating that the presence of PEtn in some positions within the LPS molecule is not required for virulence. Moreover, direct virulence trials in chickens using PEtn mutants delivered intramuscularly revealed that functional PetL or PetP were not required for VP161 or X73 to cause systemic disease in chickens. However, using in vitro antimicrobial assays we show that a functional petL gene, and therefore the presence of PEtn on lipid A, is critical for P. multocida resistance to the antimicrobial peptide, fowlicidin 1.