Nitrofurantoin is recommended as first-line therapy for the optimal treatment of uncomplicated urinary tract infections (UTIs) caused by enterococci and Escherichia coli. The mutations in nfsA and nfsB genes encoding nitroreductases can lead to nitrofurantoin resistance in gram-negative bacteria. However, the mechanisms of nitrofurantoin resistance in enterococci have not been elucidated. In this study, a total of 128 clinical isolates of Enterococcus faecium were collected from patients with UTIs and 59 (46.1%) isolates were resistant to nitrofurantoin. By analyzing the whole-genome sequences of E. faecium in the NCBI database, a type IB nitroreductase was found in all E. faecium strains and designated nrmA, representing nitroreductase in E. faecium. Nitroreductase NrmA shared 18.7% sequence identity with nitroreductase NfsB in E. coli. Different from NrmA in nitrofurantoin-susceptible E. faecium, NrmA in nitrofurantoin-resistant E. faecium had one amino acid substitution, i.e., a lysine instead of a glutamine at position 48 (Q48K mutation). This mutation was located in conserved regions, which might include the flavin mononucleotide binding site, in NfsB and other nitroreductases in gram-negative bacteria. Complementation assays of nitrofurantoin-resistant E. faecium HS17-112 showed that the nitrofurantoin minimal inhibitory concentration of the complemented strain HS17-112: pIB166-nrmA (wild type [WT]) reduced from 128 mg/L to 4 mg/L. Compared with NrmA(WT), NrmA(Q48K) showed poor catalytic efficiency for nitrofurantoin. The kcat/Km of NrmA(Q48K) for nitrofurantoin decreased from 0.122 μM-1 s-1 to 0.000042 μM-1 s-1. In conclusion, the Q48K mutation of the nitroreductase NrmA is responsible for nitrofurantoin resistance in E. faecium.