Objective To investigate the prevalence of colistin resistance and mcr-1 gene in pa-tients with bloodstream infection caused by Escherichia coli (E.coli) and Klebsiella pneumoniae (K.pneu-moniae) in Zhejiang Province. Methods A total of 869 clinical strains of the Enterobacteriaceae family, including 611 E.coli and 258 K.pneumonia strains, were isolated from patients with bloodstream infection (BSI) in Zhejiang Province from March 2014 to April 2015. Broth microdilution method and PCR were re-spectively performed to detect colistin resistance and mcr-1 gene in those stains. Susceptibilities of mcr-1-positive strains to other antibiotics were assessed by E-test. Pulsed-field gel electrophoresis(PFGE) and multilocus sequence typing(MLST) were used for molecular typing. Location of mcr-1 gene was determined by analysis of PFGE profiles of S1-digested genomic DNA and Southern blot hybridization. Plasmid transfer to E.coli recipients was investigated using filter mating test. Clinical data of the patients infected with mcr-1-positive strains was collected and analyzed. Results The minimum inhibitory concentration (MIC) values of colistin to the 869 Enterobacteriaceae strains ranged from ≤0.06 μg/ml to 16 μg/ml. Six (0.69%) E.coli strains were identified to be colistin-resistant and mcr-1-positive and the MIC values against them ranged from 8 μg/ml to 16 μg/ml. No colistin-resistant or mcr-1-positive K.pneumonia strain was identified. All mcr-1-positive strains were susceptible to carbapenems and most of them(83.33%,5/6) were suscepti-ble to tigecycline and β-lactamase inhibitor combinations tested in this study. The six mcr-1-positive strains were of different sequence types (STs) and the mcr-1 genes carried by them located on three types of plas-mids with the sizes of 33 kb,61 kb and 244.4 kb. Conclusion The prevalence of mcr-1 gene in E.coli and K.pneumonia strains isolated from patients with BSI in Zhejiang Province was relatively low, only ac-counting for 0.69% of all isolated Enterobacteriaceae strains.Those strains carrying mcr-1 gene showed low drug resistance to colistin. The mcr-1-positive strains were usually non-pathogenic clones and remained sus-ceptible to many antimicrobial agents, which was conducive to favorable outcomes. In order to clarify the clinical impact of this novel resistance gene on public health,further studies should be conducted.