Pathogenic bacteria modify the lipid A portion of their lipopolysaccharide to help evade the host innate immune response. Modification of the negatively charged phosphate groups of lipid A aids in resistance to cationic antimicrobial peptides targeting the bacterial cell surface. The lipid A of Helicobacter pylori contains a phosphoethanolamine (pEtN) unit directly linked to the 1-position of the disaccharide backbone. This is in contrast to the pEtN units found in other pathogenic Gram-negative bacteria, which are attached to the lipid A phosphate group to form a pyrophosphate linkage. This study describes two enzymes involved in the periplasmic modification of the 1-phosphate group of H. pylori lipid A. By using an in vitro assay system, we demonstrate the presence of lipid A 1-phosphatase activity in membranes of H. pylori. In an attempt to identify genes encoding possible lipid A phosphatases, we cloned four putative orthologs of Escherichia coli pgpB, the phosphatidylglycerol-phosphate phosphatase, from H. pylori 26695. One of these orthologs, Hp0021, is the structural gene for the lipid A 1-phosphatase and is required for removal of the 1-phosphate group from mature lipid A in an in vitro assay system. Heterologous expression of Hp0021 in E. coli resulted in the highly selective removal of the 1-phosphate group from E. coli lipid A, as demonstrated by mass spectrometry. We also identified the structural gene for the H. pylori lipid A pEtN transferase (Hp0022). Mass spectrometric analysis of the lipid A isolated from E. coli expressing Hp0021 and Hp0022 shows the addition of a single pEtN group at the 1-position, confirming that Hp0022 is responsible for the addition of a pEtN unit at the 1-position in H. pylori lipid A. In summary, we demonstrate that modification of the 1-phosphate group of H. pylori lipid A requires two enzymatic steps.