ObjectiveTo investigate the epidemiological characteristics of healthcareassociated infection (HAI) in neonatal intensive care units(NICUs) of tertiary firstclass hospitals in China, and provide scientific evidence for the prevention and control of neonatal HAI.  Methods17 hospitals in 9 provinces were selected for multicenter study, HAI occurred in neonates at NICUs from October 2013 to September 2014 were surveyed prospectively according to the uniform diagnostic criteria and methods. ResultsA total of 12 998 neonates were monitored, 436 (3.35%) HAI cases occurred, the total patientdays were 126 125 days, incidence of infection  was 3.46/1 000 patientdays. HAI cases rate decreased with the increase of birth weight (χ2=291.55, P＜0.001). The difference of HAI rate in neonates at different sizes of ICUs were significantly different(χ2=49.77, P＜0.001), infection case rate and infection incidence per 1 000 patientdays were both highest in NICUs with ＞30 beds; HAI case rate was highest in southwest region(10.36%), infection rate per 1 000 patientdays was the highest in southern China(10.52‰). Lower respiratory tract and bloodstream were the main infection sites; the major pathogens for infection were  Klebsiella pneumoniae, Escherichia coli, and  Acinetobacter baumannii.ConclusionThe incidences of HAI in NICUs are different among neonates of different birth weight, at different sizes of ICUs, and at ICUs of different regions, surveillance should be strengthened, prevention and control measures should implemented according to infection characteristics.