Background: Enterococcus faecium is an important nosocomial pathogen and has developed resistance to multiple antibiotics, such as vancomycin. Nitrofurantoin is active against E. faecium and E. faecalis, including vanA- and vanB-positive isolates. Objectives: To investigate the role of rpoB mutation in E. faecium. Methods: The growth rate and reactive oxygen species (ROS) levels were determined. The proteomic profiles of E. faecium B42 and its Rifr mutant B42-R7 were also compared. Complement experiments were performed to elucidate the role of nitroreductase in nitrofurantoin resistance. Results: The Rifr mutant (B42-R7, rpoB H489D) of E. faecium had a lower growth rate and higher ROS levels. Overexpression of nitroreductase in E. faecium B42-R7 was observed in proteomic analyses. Enhanced expression of nitroreductase in Escherichia coli increased the sensitivity to nitrofurantoin. Conclusions: The rpoB mutation in E. faecium not only altered the susceptibility to rifampin, but also affected global protein expression, including nitroreductase. Nitroreductase expression in E. faecium B42-R7 might play a role in nitrofurantoin resistance.