Abstract: 　Objective　To compare the clinical characteristics and drug susceptibility of neonatal early-onset sepsis (EOS) and late-onset sepsis (LOS) caused by Escherichia coli. Methods　The clinical data of 137 neonates with Escherichia coli septicemia from September 2000 to May 2017 were retrospectively analyzed. All neonates were divided into EOS group (onset within 72 hours after birth) and LOS group (onset over 72 hours after birth) according to the time of sepsis, and the clinical data of two groups were compared. Results　There were 74 cases in EOS group and 63 cases in LOS group. Compared with LOS group, infants in EOS group had higher incidence of premature rupture of membranes (21.6% vs. 7.9%), and was more likely to have coagulation dysfunction (39.2% vs. 17.5%), intracranial hemorrhage (37.8% vs. 19%), respiratory failure (29.7% vs. 14.3%), and hypoglycemia (16.2% vs. 4.8%). The differences were statistically significant (all P<0.05). There was no difference in sensitivity to third-generation cephalosporins, aminoglycosides, quinolones and carbapenems antibiotics between the two groups (all P>0.05). The rate of Escherichia coli resistant to ampicillin was up to 70%, and the rate to cephalosporins except ceftazidime was also high. While the rate of Escherichia coli sensitive to antibiotics containing β-lactamase inhibitors was 71.8%~90.7%, the rate to quinolones and aminoglycosides was over 70%, and the rate to carbopenems was up to 97%. Conclusion　Neonates with early-onset Escherichia coli sepsis had more complications than those with late-onset sepsis. There was no difference in drug sensitivity between early-onset and late-onset neonatal Escherichia coli sepsis. Antibiotics containing β-lactamase inhibitors should be the first choice in treatment of Escherichia coli sepsis, and carbapenems could be used if its efficacy is poor.