ABSTRACT Salmonella enterica serovar Typhimurium utilizes a type III secretion system (TTSS) encoded on Salmonella pathogenicity island-2 (SPI2) to promote intracellular replication during infection, but little is known about the molecular function of SPI2-translocated effectors and how they contribute to this process. SseJ is a SPI2 TTSS effector protein that is homologous to enzymes called glycerophospholipid-cholesterol acyltransferases and, following translocation, localizes to the Salmonella -containing vacuole and Salmonella- induced filaments. Full virulence requires SseJ, as sseJ null mutants exhibit decreased replication in cultured cells and host tissues. This work demonstrates that SseJ is an enzyme with deacylase activity in vitro and identifies three active-site residues. Catalytic SseJ mutants display wild-type translocation and subcellular localization but fail to complement the virulence defect of an sseJ null mutant. In contrast to the wild type, SseJ catalytic mutants fail to down regulate Salmonella -induced filament formation and fail to restore the sifA null mutant phenotype of loss of phagosomal membrane to sifA sseJ null double mutants, suggesting that wild-type SseJ modifies the vacuolar membrane. This is the first demonstration of an enzymatic activity for a SPI2 effector protein and provides support for the hypothesis that the deacylation of lipids on the Salmonella -containing vacuole membrane is important to bacterial pathogenesis.