Abstract: Objective To investigate the clinical characteristics and drug resistance in patients with bloodstream infection (BSI) due to K. pneumonia or E. coli. Methods We conducted a retrospective analysis on patients with BSI due to K. pneumoniae or E. coli from January 2013 to December 2015 in Chinese PLA General Hospital. Data were collected on underlying diseases, source of infection, production of extended spectrum β-lactamase (ESBL), and drug sensitive test. Results Four hundred and thirtysix cases of BSI were enrolled in our study, with 282(64.7%) E. coli BSI and 154(35.3%) K. pneumoniae BSI. In-hospital 30-day mortality of patients were higher in K. pneumoniae (20.1%) than that in E. coli (9.2%) (P=0.002). The most common sources of infection were pancreaticobiliary infection (30.1%) and urinary tract infection (20.2%) due to E.coli, and lung infection (37.7%) due to K. pneumoniae. ESBL was isolated from 56.8% of E. coli BSI and 24.8% of K. pneumoniae BSI, respectively (P< 0.001). Compared with K. pneumoniae, the resistance rates to fluoroquinolone were higher in E. coli (63%-64.8%). Carbapenem-resistant Enterobacteriaceae (CRE) strains accounted for an overall rate of 11%-12.9%, in which the resistance rates were up to 26.8%-30.7% for K. pneumoniae, and 2.2%-2.9% for E. coli. The resistance rate to amikacin (9.3%) was the lowest in the two strains. Conclusion The clinical characteristics and prognosis of BSI caused by E. coli and K. pneumoniae are significantly different. Both strains show high resistance rates to major antibiotics with Carbapenem-resistant rates of 26.8%-30.7% in K. pneumoniae. Rational use of antibiotics and hospital infection control should be strengthened in clinical practice.