Vancomycin-resistant enterococci are important nosocomial pathogens that require rapid and accurate detection for infection control. Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS) has begun to be used in many clinical laboratories because it is a rapid, simple and inexpensive method for identifying micro-organisms. In this study, the performance of MALDI-TOF/MS to differentiate vanA-positive Enterococcus faecium (VPEF) from vanA-negative E. faecium (VNEF) was evaluated. A total of 61 VPEF isolates collected during regional surveillance in Kyoto (Japan) and 71 VNEF isolates collected from bacteraemia patients were analysed using MALDI-TOF/MS with three ClinProTools models. All of the isolates were correctly identified as E. faecium using the MALDI Biotyper system. To discriminate between VPEF and VNEF, all three ClinProTools models yielded >90% recognition capability (basic sensitivity) and cross-validation (reliability of the models); the genetic algorithm model exhibited the highest performance (99.18% and 92.40%, respectively). The high detection performance of MALDI-TOF/MS for VPEF offers the potential for routine laboratory use.