Background: Contaminated parenteral nutrition (PN) is an important source of infection in neonates. Many organisms have been reported to cause contamination resulting in outbreaks in intensive care units. The aim of the present study was to investigate an outbreak caused by Serratia marcescens in a neonatal intensive care unit (NICU). Methods: This was a descriptive study of an outbreak of sepsis in an NICU of a university teaching hospital. The outbreak was detected in seven patients from 10 to 12 December 2005 following the administration of PN. Extensive environmental samplings for culture were performed. The clonal relationship among isolates was tested using pulsed-field gel electrophoresis, random amplification of polymorphic DNA-polymerase chain reaction and plasmid DNA typing. Results: Serratia marcescens was found in blood cultures from infected newborns and from in-use PN solutions. Gestational age of the seven babies ranged from 28 to 34 weeks (median, 32 weeks), birthweight ranged from 1000 g to 2190 g (median, 1469 g), and postnatal age ranged from 8 to 22 days. The mortality rate was 14.3%. All these strains of S. marcescens had the same antibiotic susceptibility pattern and the same genomic DNA profile. Plasmid typing, as well as RAPD-PCR showed that all isolates had the same profile. Conclusion: The source of the nosocomial sepsis in seven neonates was the PN solution. Contamination may occur during storage or repeated handling during PN preparation. © 2010 Japan Pediatric Society.