In the major human pathogen Klebsiella pneumoniae, MgrB inactivation by disruptive insertion sequence (IS) elements and mutations leading to early termination are known to play an important role in polymyxin resistance. In this study, we examined a collection of invasive blaKPC-2-producing K. pneumoniae isolates belonging to the high-risk clone sequence type 258 (ST258) displaying high rates of resistance to many antimicrobials, including polymyxins. We identified a deleterious substitution (W20S) in MgrB and confirmed by genetic complementation analysis that this variant was inactive, leading to increased polymyxin B and colistin MICs.