The aim of this work was to study the phenotypic and molecular characterization of a novel plasmid-mediated AmpC beta-lactamase from Escherichia coli E384. Conjugation experiments, isoelectric focusing, pulsed-field gel electrophoresis, plasmid profiling, and Southern blot as well as PCR, sequencing techniques, and susceptibility testing were carried out to investigate the underlying mechanism of resistance. The kinetic parameters were determined to characterize the novel enzyme. MIR-4 beta-lactamase, pI 8.2, is a novel variant with four substitutions of amino acids compared with the sequence of MIR-1. E. coli E384 displays resistance to eight beta-lactam antimicrobial agents and three fluoroquinolones. The minimal inhibitory concentrations of beta-lactam in combination with beta-lactamase inhibitors show no significant synergy. Kinetic parameters suggest that the novel enzyme effectively hydrolyzes broad-spectrum beta-lactams. The same hybridization signal was detectable only in the 54-kb plasmid band that hybridized with the bla (CTX-M)- and bla (ampC)-specific probes. This is the first description of a plasmid-mediated MIR-4 enzyme in China. This study illustrates the importance of molecular surveillance in tracking AmpC-producing strains at general hospitals and emphasizes the need for epidemiological monitoring.