SifA is a Salmonella typhimurium effector protein that is translocated across the membrane of the Salmonella-containing vacuole by the Salmonella pathogenicity island 2-encoded type III secretion system. SifA is necessary for the formation of Salmonella-induced filaments and for the maintenance of the vacuolar membrane enclosing the pathogen. We have investigated the role of the C-terminal hexapeptide of SifA as a potential site for membrane anchoring. An S. typhimurium strain carrying a deletion of the sequence encoding this hexapeptide (sifA Delta 6) was found to be attenuated for systemic virulence in mice. In mouse macrophages, sifA Delta 6 mutant bacteria displayed a reduced association with vacuolar markers, similar to that of sifA null mutant bacteria, and exhibited a dramatic replication defect. Expression of SifA in epithelial cells results in the mobilization of lysosomal glycoproteins in large vesicular structures and Sif-like tubules. This process requires the presence of the C-terminal hexapeptide domain of SifA. Ectopic expression of truncated or mutated versions of SifA affecting the C-terminal hexapeptide revealed a strong correlation between the membrane binding capability and the biological activity of the protein. Finally, the eleven C-terminal residues of SifA are shown to be sufficient to target the Aequorea green fluorescent protein to membranes. Altogether, our results indicate that membrane anchoring of SifA requires its C-terminal hexapeptide domain, which is important for the biological function of this bacterial effector.