Objetive To explore bacteriological changes and susceptible factor of nosocomial infection in neonatal intensive care unit (NICU). Methods The clinical data from 5543 hospitalized neonates during January 2010 to December 2015 were retrospectively analyzed. Results Nosocomial infection rate during the study period was 8.75%. The most common pathogen of nosocomial infection was Staphylococcus epidermidis, followed by Escherichia coli and Klebsiella pneumoniae. Respiratory infection accounted for 37.73% and blood infection 37.53%. 36.74% blood samples were tested to be positive, and 32.67% sputum were positive. In the first three years, the main pathogens of nosocomial infection were Candida albicans, Klebsiella pneumonia, and Staphylococcus aureus; in the last three years, the main pathogens were Staphylococcus aureus, Escherichia coli, and Klebsiella pneumoniae. The susceptible factors of nosocomial infection in NICU were gestational age?