Staphylococcus aureus sortase A cleaves surface protein precursors bearing C-terminal LPXTG motif sorting signals between the threonine and glycine residues. Using lipid II precursor as cosubstrate, sortase A catalyzes the amide linkage between the carboxyl group of threonine and the amino group of pentaglycine cross-bridges, thereby tethering C-terminal ends of surface proteins to the bacterial cell wall envelope. Staphylococcal sortase B also anchors its only known substrate, the IsdC precursor with a C-terminal NPQTN motif sorting signal, to the cell wall envelope. Herein, we determined the cell wall anchor structure of IsdC. The sorting signal of IsdC is cleaved between threonine and asparagine of the NPQTN motif, and the carboxyl group of threonine is amide-linked to the amino group of pentaglycine crossbridges. In contrast to sortase A substrates, the anchor structure of IsdC displays shorter glycan strands and significantly less cell wall cross-linking. A model is proposed whereby sortases A and B recognize unique features of sorting signals and peptidoglycan substrates to deposit proteins with distinct topologies in the cell wall envelope.