Objectives: To investigate characteristics of nine carbapenem-non-susceptible (CP-NS) Escherichia coli isolates collected between 1999 and 2005 at a Taiwanese university hospital. Methods: Genetic relatedness was analysed by PFGE. beta-Lactamases were characterized by PCR and isoelectric focusing. Outer membrane proteins and transcripts were investigated by SDS-PAGE and northern blotting. Cloning experiments were performed to investigate the role of membrane permeability in carbapenem non-susceptibility. Results: The nine CP-NS isolates were found to produce the CMY-2 AmpC enzyme (n = 8), the CTX-M-14-type extended-spectrum beta-lactamase (ESBL) (n = 1), the SHV-12 ESBL (n = 1) and the IMP-8-type metallo-beta-lactamase (n = 1) alone or in combination. All CP-NS isolates revealed a decrease in the transcription and protein expression of ompC, and susceptibility to carbapenems was restored in one isolate by introducing the cloned ompC gene. PFGE revealed genetic diversity among the nine isolates. All patients with the CP-NS isolates had been treated with carbapenems (six patients) and/or extended-spectrum cephalosporins (five patients) before isolation. Conclusions: Our study suggests that the decreased susceptibility to carbapenems in E. coli in the hospital might arise by the stepwise accumulations of multiple drug-resistance determinants in different clones.