The objective of this study was to better understand the in vitro activity of flomoxef against clinical extended-spectrum p-lactamase (ESBL)-producing Enterobacteriaceae. A total of 401 ESBL-producing isolates, including 196 Escherichia coli, 124 Klebsiella pneumoniae and 81 Proteus mirabilis, were collected consecutively from 21 hospitals in China in 2013. Minimum inhibitory concentrations (MICs) were determined by broth microdilution methods. Phenotypic identification of ESBL production was detected as recommended by the Clinical and Laboratory Standards Institute (CLSI). ESBL genes were detected by PCR and sequencing. Flomoxef, doripenem, meropenem, ertapenem, cefmetazole and piperacillin/tazobactam exhibited good activity against ESBL-producing isolates, with susceptibility rates >90%. Tigecycline showed good activity against E. coli and K. pneumoniae (100% and 97.6%, respectively). Cefotaxime and cefepime showed very low activities against ESBL-producing isolates, with susceptibility rates of 0-0.8% and 1.0-13.6%, respectively. bla(CTXM) were the major ESBL genes, with occurrence in 99.5% of E. coli, 91.1% of K. pneumoniae and 97.5% of P. mirabilis. bla(CTX-M-14) was the predominant ESBL gene, detected in 46.9% (188/401) of the isolates, followed by bla(CTX-M-15) (2 1.4%), bla(CTX-M-55) (1 7.2%), bla(CTX-M-65) (1 2.7%) and bla(CTX-M-3) (6.7%). Flomoxef exhibited excellent activity against the different CTX-M-type ESBL-producing isolates, with MIC50 and MIC90 values of 0.064-0.125 mu g/mL and 0.25-0.5 mu g/mL, respectively. Against the isolates solely producing CTX-M-14, -15, -55, -3 or -65, flomoxef showed susceptibility rates of 98.6%, 98.0%, 98.1%, 100.0% and 97.4%, respectively. In conclusion, flomoxef showed good activity against ESBL-producing Enterobacteriaceae and may be a choice to treat infections caused by these isolates in China. (C) 2015 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.