Pasteurella multocida is the causative agent of a number of animal diseases, including fowl cholera. P. multocida strains express two lipopolysaccharide (LPS) glycoforms simultaneously (A and B) that differ only in their inner core structure. Glycoform A contains a single 3-deoxy-D-manno-octulosonic acid (Kdo) residue that is phosphorylated by the Kdo kinase, KdkA, whereas glycoform B contains two un-phosphorylated Kdo residues. Previously we have shown that P. multocida mutants lacking the heptosyltransferase, HptA, produce full-length glycoform B LPS and a large amount of truncated glycoform A LPS, as they cannot add heptose to the glycoform A inner core. These hptA mutants were attenuated in chickens because the truncated LPS made them vulnerable to host defence mechanisms including antimicrobial peptides. However, here we show that birds inoculated with high doses of the hptA mutant developed fowl cholera and P. multocida recovered from diseased birds no longer expressed truncated LPS. Sequencing analysis revealed that in vivo isolates had mutations in kdkA, thereby suppressing the production of glycoform A LPS. Interestingly, a number of the spontaneous KdkA mutant strains produced KdkA with a single amino acid substitution (A112V, R123P, H168Y or D193N). LPS structural analysis showed that complementation of a P. multocida kdkA mutant with wild-type kdkA restored expression of glycoform A to wild-type levels, whereas complementation with any of the mutated kdkA genes did not. We conclude that in P. multocida KdkA, the amino acids A112, R123, H168 and D193 are critical for Kdo kinase function and therefore for glycoform A LPS assembly.