Carbapenems are beta-lactam antibiotics commonly used to treat severe infections by bacteria resistant to multiple antibiotic classes. The emergence and global spread of Klebsiella pneumoniae carbapenemase (KPC) enzymes among Enterobacteriaceae, which hydrolyse all beta-lactams including last line carbapenem antibiotics and are also resistant to beta-lactamase inhibitors, is a major threat to global public health. Consequently, carbapenemase-producing bacteria are listed by the WHO as ‘Critical Priority’ pathogens for new antimicrobial development. The KPC enzyme contains a disulfide bridge between Cys69 and Cys238, located close to the active site. However, the role of this disulfide bridge in maintaining KPC structural stability or enzyme activity remains unresolved. Here we report that disrupting the disulfide bridge in KPC by mutating the Cys69 residue to Ser, renders the enzyme less stable and less active for beta-lactam hydrolysis. In Escherichia coli the KPC C69S mutant confers reduced susceptibility to beta-lactams, including carbapenems. In addition, KPC-producing clinical E. coli isolates lacking the DsbA enzyme, which catalyses disulfide bond formation, are also significantly more susceptible to carbapenem antibiotics. Inhibition of DsbA in KPC-producing isolates with a small molecule inhibitor restores their clinical susceptibility to several carbapenem antibiotics. Our findings demonstrate the essential role of the disulfide bridge in the functional biogenesis of the most widespread class A carbapenemase and provide significant proof of principle for DsbA inhibition as an innovative antibiotic adjuvant strategy with a new mode of action for beta-lactamase inhibition.