ObjectiveTo understand the occurrence and risk factors of healthcareassociated infection(HAI) in a neonatal intensive care unit(NICU). MethodsNeonates who were admitted to the NICU of a hospital from January 2012 to December 2014 were investigated retrospectively, risk factors for HAI were performed univariate analysis. ResultsA total of 760 neonates were included in the investigation, 198 neonates developed 259 times of HAI, incidence of HAI was 26.05%，case incidence of HAI was 34.08%，incidence of HAI per 1 000 patient days was 9.50‰； the main infection site was lower respiratory tract （n=92, 35.52%）； among 259 cases of HAI, 172 strains of pathogenic bacteria were isolated, the major pathogen was Acinetobacter spp.(n=40, 23.26%）; incidence of HAI was high in neonates with gestational age ≤32 weeks, birth weight≤1 500 g, length of hospital stay≥10 days, duration of antimicrobial use≥10 days, mechanical ventilation, deep venous catheterization, and feeding intolerance, difference was statistically significant (all P<0.001). ConclusionIncidence of HAI in NICU is high, effective prevention and control measures should be formulated according to its risk factors, so as to reduce the occurrence of HAI in neonates.