UNLABELLED: Cell wall-anchored surface proteins of Gram-positive bacteria, harboring a highly conserved YSIRK/G-S signal peptide (SP IMPORTANCE: Surface proteins containing a YSIRK/G-S-positive signal peptide are widely distributed in Gram-positive bacteria and play essential roles in bacterial pathogenesis. They are highly expressed proteins that are enriched at the septum during cell division. The biogenesis of these proteins is coordinated with cell cycle and LTA synthesis. The current study identified the staphylococcal signal peptidase SpsB as a key determinant in regulating surface protein septal trafficking. Furthermore, this study highlights the novel functions of SpsB in coordinating LtaS-mediated LTA production and regulating staphylococcal cell cycle. As SpsB, YSIRK+ proteins, and LTA synthesis are widely distributed and conserved, the mechanisms identified here may be shared across Gram-positive bacteria.