Mupirocin is a topical antibiotic used for the treatment of skin infections and the eradication of methicillin-resistant Staphylococcus aureus carriage. It inhibits bacterial protein synthesis by interfering with isoleucyl-tRNA synthetase activity. High-level mupirocin resistance (MIC of ≥ 512 μg/ml) is mediated by the expression of mupA (ileS2), which encodes an alternate isoleucyl-tRNA synthetase. In this study, we describe high-level mupirocin resistance mediated by a novel locus, mupB. The mupB gene (3,102 bp) shares 65.5% sequence identity with mupA but only 45.5% identity with ileS. The deduced MupB protein shares 58.1% identity (72.3% similarity) and 25.4% identity (41.8% similarity) with MupA and IleS, respectively. Despite this limited homology, MupB contains conserved motifs found in class I tRNA synthetases. Attempts to transfer high-level mupirocin resistance via conjugation or transformation (using plasmid extracts from an mupB-containing strain) were unsuccessful. However, by cloning the mupB gene into a shuttle vector, it was possible to transfer the resistance phenotype to susceptible S. aureus by electroporation, proving that mupB was responsible for the high-level mupirocin resistance. Further studies need to be done to determine the prevalence of mupB and to understand risk factors and outcomes associated with resistance mediated by this gene.