The lipid A portion of lipopolysaccharide, the major component of the outer leaflet of the outer membrane of Gram-negative bacteria, is toxic to humans. Modification of lipid A by enzymes often reduces its toxicity. The outer-membrane protein LpxR from Salmonella typhimurium is a lipid A-modifying enzyme. It removes the 3′-acyloxyacyl moiety of the lipid A portion of lipopolysaccharide in a Ca ²⁺ -dependent manner. Here, we present the crystal structure of S. typhimurium LpxR, crystallized in the presence of zinc ions. The structure, a 12-stranded β-barrel, reveals that the active site is located between the barrel wall and an α-helix formed by an extracellular loop. Based on site-directed mutagenesis and modeling of a substrate on the active site, we propose a catalytic mechanism similar to that of phospholipase A2, in which a Ca ²⁺ forms the oxyanion hole and a histidine activates a water molecule (or a cascade of two water molecules) that subsequently attacks the carbonyl oxygen of the scissile bond.