Abstract: Objective To investigate the composition of nosocomial infection sites and pathogens changes in hospitalized children in a children's hospital and provide scientific basis for prevention and control of nosocomial infections. Methods Hospitalized children diagnosed with nosocomial infection from January 1, 2012 to December 31, 2017 were selected as the study subjects. Specimens were collected for pathogen detection. The composition of nosocomial infection sites and pathogens changes were analyzed, comparing 2012-2014 with 2015-2017. Results The total number of children diagnosed with nosocomial infection was 1 991 and the number of nosocomial infections was 2 022. There were 1 212(60.9%) boys, and the age of the children was 2.0(0.6,5.0) years(1 d to 17 years). There were no significant differences in gender and seasons in children with nosocomial infection between 2012-2014 with 2015-2017 (P was 0.618 and 0.145, respectively), while the difference in age distribution was statistically significant (P< 0.001). ①The top four infection sites were lower respiratory tract (28.8%), gastrointestinal tract (25.7%), upper respiratory tract (25.2%) and bloodstream infection (10.4%). Comparing 2012-2014 with 2015-2017, the compositions of lower respiratory tract and bloodstream infections were significantly increased (P=0.016 and 0012, respectively). The most frequent sites of nosocomial infection were bloodstream infection in Neonatal Department(49.1%), lower respiratory tract infection in PICU (69.6%), respiratory infection in general wards (58.7%), while infection rate in the central nervous system, urinary tract, and surgical site in Department of Pediatric Surgery was higher than those in Neonatal Department, PICU and general wards. ②The rate of microbiological examination was 81.8%(1 655/2 022), and the positive rate was 49.3%(821/1 667). The most frequent pathogens detected were gram-negative bacteria (39.4%) and virus (34.8%), followed by gram-positive bacteria (17.9%) and fungi (6.9%). Comparing 2012-2014 with 2015-2017, the composition of gram-negative bacteria was decreased (45.2% vs 34.6%, P=0.002), while that of gram-positive bacteria was increased (13.8% vs 213%, P=0.005). Klebsiella pneumoniae was the top pathogen to be detected(14.0%), which mainly caused the hospital's lower respiratory tract (44.0%) and bloodstream (44.0%) infections. Acinetobacter baumannii ranked the second (13.4%), and up to 75% of the strains caused lower respiratory tract infections. In addition, about 2/3 of enterococci caused bloodstream infections. Gastrointestinal infections were mainly caused by rotavirus-infected diarrhea. Conclusion The composition of nosocomial infection sites in different departments is different. PICU and Neonatal Department need to pay attention to hospital-acquired pneumonia and bloodstream infections. Invasive operations should be reduced or avoided and the antimicrobial drugs should be used rationally to minimize the occurrence and spread of drug-resistant bacteria.