Abstract: Objective　To investigate the effect of standardized usage of antibiotics and hand hygiene on carbapenem-resistant Enterobacteriaceae (CRE) infection in neonates. Methods According to the situation of standardized management of antibiotics in our hospital, the study period, from January 2011 to December 2013, was divided into pre-intervention, transition and post-intervention. Retrospective study was employed to analyze the antibiotics use density (AUD), the hand hygiene compliance rate among the medical staff, the positive rate of CRE and the nosocomial infection percentage of the neonatology department. Results From pre-intervention to post-intervention period, the AUD was decreased from 12.93 DDDs to 8.10 DDDs; hand hygiene compliance rate of medical staff was increased from 53.49% to 83.33%; the detection rate of CRE was decreased from 3.71% to 2.27%. The differences were all significant among different periods (P<0.01). The nosocomial infection percentage decreased from 0.22% to 0.17% and there was no significant difference (P=0.441). Conclusions Standardized usage of antibiotics and hand hygiene can be served as economical yet effective approaches to controlling CRE infection in neonates.