Carbapenems are first-line agents for the treatment of serious nosocomial infections caused by multidrug-resistant Enterobacteriaceae. However, resistance to carbapenems has increased dramatically among Enterobacteriaceae in our hospital. In this study, we report clonal dissemination caused by carbapenem-resistant Enterobacter aerogenes (CREA). In 2011, CREA was identified from 12 patients admitted to the neurosurgical ward. All 12 clinical isolates were non-susceptible to cefotaxime, ceftazidime, cefoxitin, ertapenem, imipenem or meropenem. All isolates carried the gene encoding Klebsiella pneumoniae carbapenemase-2 (KPC-2), except for the isolate E4. However, a remarkably lower expression level of the porin OmpF was detected in the non-KPC-2-producing isolate E4 on SDS-PAGE compared with the carbapenem-susceptible isolate. Epidemiological and molecular investigations showed that a single E. aerogenes strain (PFGE type A), including seven KPC-2-producing clinical isolates, was primarily responsible for the first isolation and subsequent dissemination. In a case-control study, we identified risk factors for infection/colonization with CREA. Mechanical ventilation, the changing of sickbeds and previous use of broad-spectrum antibiotics were identified as potential risk factors. Our findings suggest that further studies should focus on judicious use of available antibiotics, implementation of active antibiotic resistance surveillance and strict implementation of infection-control measures to avoid the rapid spread or clonal dissemination caused by carbapenem-resistant Enterobacteriaceae in healthcare facilities.