Staphylococcus aureus is a bacterial pathogen that produces and exports many virulence factors that cause diseases in human. PrsA, a membrane-bound foldase, is expressed ubiquitously in Gram-positive bacteria and required for the folding of exported proteins into a stable and active structure. To understand the involvement of PrsA in post-translocational protein folding in S. aureus, a PrsA-deficient mutant of S. aureus HG001 was constructed. Using isobaric tags for relative and absolute quantification (iTRAQ)-based mass spectrometry analyses, the exoproteomes of PrsA mutant and wild type S. aureus were comparatively profiled, and 163 cell wall-associated proteins and 67 exoproteins with altered levels have been identified in the PrsA-deficient mutant. Bioinformatics analyses further reveal that prsA deletion altered the amounts of proteins that are potentially involved in the regulation of cell surface properties and bacterial pathogenesis. To determine the relevancy of our findings, we investigated the functional consequence of prsA deletion in S. aureus. PrsA deficiency can enhance bacterial auto-aggregation and increase the adhesion ability of S. aureus to human lung epithelial cells. Moreover, mice infected with PrsA-deficient S. aureus had a better survival rate compared with those infected with the wild-type S. aureus. Collectively, our findings reveal that PrsA is required for the post-translocational folding of numerous exported proteins and critically affects the cell surface properties and pathogenesis of S. aureus. This article is protected by copyright. All rights reserved.