Sortases cleave short peptide motif sequences at the C-terminal end of secreted surface protein precursors and either attach these polypeptides to the peptidoglycan of Gram-positive bacteria or promote their assembly into pilus structures that are also attached to peptidoglycan. Sortase A, the enzyme first identified in the human pathogen Staphylococcus aureus, binds LPXTG motif sorting signals, cleaves between threonine (T) and glycine (G) residues, and forms an acyl enzyme between its active-site cysteine thiol and the carboxyl group of threonine (T). Sortase A acyl enzyme is relieved by the nucleophilic attack of the cross bridge amino group within lipid II, thereby generating surface protein linked to peptidoglycan precursor. Such products are subsequently incorporated into the cell wall envelope by enzymes of the peptidoglycan synthesis pathway. Surface proteins linked to peptidoglycan may be released from the bacterial envelope to diffuse into host tissues and fulfill specific biological functions. S. aureus sortase A is essential for host colonization and for the pathogenesis of invasive diseases. Staphylococcal sortase-anchored surface proteins fulfill key functions during the infectious process, and vaccine-induced antibodies targeting surface proteins may provide protection against S. aureus. Alternatively, small-molecule inhibitors of sortase may be useful agents for the prevention of S. aureus colonization and invasive disease.