ABSTRACT Penicillin-binding protein 4 (PBP4), a nonessential, low-molecular-weight penicillin-binding protein of Staphylococcus aureus , has been implicated in low-level resistance to β-lactam antibiotics, although the mechanism is unknown. Mutations in PBP4 and its promoter were identified in a laboratory-generated mutant strain, CRB, which expresses high-level resistance to β-lactams, including resistance to the new-generation cephalosporins active against methicillin-resistant strains of S. aureus . These mutations did not appreciably alter the β-lactam antibiotic binding affinity of purified recombinant mutant PBP4 compared to that of wild-type PBP4. Compared to the susceptible parent strain, COLnex, the CRB strain produces a highly cross-linked cell wall peptidoglycan, indicative of increased transpeptidase activity. The pbp4 promoter mutation of CRB was associated with greatly increased amounts of PBP4 in membranes compared to those in the COLnex parent. Replacement of the native promoter of COLnex with the mutant promoter of CRB resulted in increased amounts of PBP4 in membranes and a highly cross-linked cell wall. PBP4 can be repurposed to provide essential transpeptidase activity in vivo and confer high-level resistance to β-lactam antibiotics, such as ceftobiprole and ceftaroline.