We have characterized the structure of lipid A in the food-borne pathogen Cronobacter sakazakii BAA-894. Lipid A molecules were purified by mild acid hydrolysis, DEAE–cellulose anion exchange chromatography and preparative thin-layer chromatography. Its structure was analyzed by electrospray ionization/mass spectrometry. Two lipid A species have been found in C. sakazakii BAA-894. The majority has a structure of disaccharide of glucosamine, phosphorylated at the 1- and 4′-positions, and acylated with primary 3-hydroxy myristoyl chains at the 2-, 3-, 2′- and 3′-positions and secondary myristoyl residues at the 2′- and 3′-positions. The other contains a secondary lauroyl chain at the 2′-position in place of the myristoyl residue.