Objective To explore risk factors for neonatal carbapenem-resistant Enterobacteriaceae (CRE) infection, and provide evidence for prevention and control of neonatal CRE infection and rational use of antimicrobial agents. Methods 68 neonates with CRE infection in a neonatal ward of Wuhan Children's Hospital from 2016 to 2018 were chosen as CRE group, 50 neonates with carbapenem-sensitive Enterobacteriaceae (CSE) infection in the same ward during the same period were randomly selected as CSE group, retrospective case-control study was conducted, risk factors for CRE infection were analyzed. Results There were 68 neonates in CRE group, including 60 cases (88.24%) of Klebsiella pneumoniae infection, 7 cases (10.29%) of Escherichia coli infection and 1 case (1.47%) of Klebsiella oxytoca infection. There were 38 cases (55.88%) of healthcare-associated infection(HAI) in CRE group and 6 cases (12.00%) of HAI in CSE group, difference between two groups of neonates was significant (P<0.05). Lower respiratory tract was the main infection site in both groups. Univariate analysis showed that small gestational age, low birth weight, neonatal respiratory distress syndrome, endotracheal intubation, mechanical ventilation time, peripherally inserted central catheterization (PICC), antimicrobial use time, types of used antimicrobial agents ≥ 3 kinds, use history of carbapenems, use history of glucocorticoid, long hospital stay were all risk factors for neonatal CRE infection. Multivariate regression analysis showed that low birth weight, duration of mechanical ventilation and history of carbapenem use were independent risk factors for CRE infection. Conclusion The main neonatal CRE infection is HAI, comprehensive prevention and control measures should be formulated according to the risk factors of neonatal CRE infection, so as to reduce the occurrence and spread of CRE infection.