The objective of this investigation was to evaluate the association between colistin consumption and the isolation of intrinsically resistant to colistin Enterobacteriaceae (IRCE) in a university hospital in Crete, Greece. The database of the microbiological laboratory was reviewed retrospectively during 2006-2010. All positive cultures for IRCE were retrieved. We assessed the total consumption of colistin in medical, surgical, and intensive care units (ICUs). A total of 1,304 single-patient IRCE isolates were recorded. Of these, 466 (35.7%) were hospital-acquired, while 838 (64.3%) were community-acquired. Proteus spp. accounted for 72% of them, Serratia spp. for 16.6%, Morganella morganii for 8.4%, and Providencia spp. for 3%. Urine (44.8%), pus (20.4%), and lower respiratory tract specimens (12.8%) accounted for the majority of specimens. IRCE isolated during the first half (2006 to 1st semester of 2008) and second half (2nd semester of 2008 to 2010) of the study period accounted for 5.8% and 7.4% of Gram-negative isolates, respectively (p < 0.001). Colistin consumption was not different in the two periods in the hospital, but in the ICU, it was higher in the second half of the study period (p = 0.013). Colistin consumption was associated with the isolation of hospital-acquired IRCE (p = 0.037); a trend was noted between colistin consumption and the isolation of IRCE in the ICU (p = 0.057). In this study, colistin consumption was associated with the isolation of hospital-acquired IRCE. The use of colistin increased in the ICU during the study period. Prudent use of colistin is essential for the prevention of nosocomial outbreaks due to resistant IRCE.