Objective To analyze the influencing factors for intestinal colonization and secondary infection of carbapenem-resistant Klebsiella pneumoniae (CRKP) in neonates, and provide a basis for formulating prevention and control strategies for CRKP infection. Methods Neonates who were admitted to the neonatal ward of a hospital from January 2021 to October 2022 were selected as the study subjects, and the first screening of CRKP was conducted within 48 hours after admission. In addition, active anal swab screening for carbapenem-resistant Ente-robacterales (CRE) was performed weekly during hospitalization, and the infection status of CRKP strains was monitored. Clinical data of neonates in the colonization group, non-colonization group, and infection group were analyzed. Intestinal colonized strains and the non-repetitive CRKP strains isolated from clinical specimens of neonates with secondary infection after colonization were performed carbapenemase gene detection, multilocus sequence ty-ping (MLST) and pulsed-field gel electrophoresis (PFGE) analysis. Results A total of 1 438 neonates were actively screened for CRE, 174 were CRKP positive, CRKP colonization rate was 12.1%. Among 174 neonates, 35 were with secondary infection, with the incidence of 20.1%. The independent risk factors for neonatal CRKP intestinal colonization were cesarean section (OR=2.050, 95%CI: 1.200-3.504, P=0.009), use of cephalosporins (OR=1.889, 95%CI: 1.086-3.288, P=0.024), nasogastric tube feeding (OR=2.317, 95%CI: 1.155-4.647, P=0.018). Protective factors were breast-feeding (OR=0.506, 95%CI: 0.284-0.901, P=0.021), oral probiotics (OR=0.307, 95%CI: 0.147-0.643, P=0.002), and enema (OR=0.334, 95%CI: 0.171-0.656, P=0.001). Independent risk factors for secondary infection after intestinal colonization of neonatal CRKP were carbapenem antibiotic use (OR=19.869, 95%CI: 1.778-222.029, P=0.015) and prolonged hospital stay(OR=1.118, 95%CI: 1.082-1.157, P < 0.001). The detection results of drug resistance genes showed that carbapenemase-producing genes of CRKP strains were all bla_KPC-2, all belonged to type ST11. Homologous analysis showed that intestinal CRKP colonization was highly homologous with the secondary infection strains after colonization. Conclusion CRKP intestinal colonization during neonatal hospitalization may increase the risk of CRKP infection. Risk and protective factors of neonatal intestinal colonization and secondary infections after colonization should be paid attention, and corresponding preventive and control measures should be taken, so as to reduce the occurrence and transmission CRKP healthcare-associated infection.