Objectives:To determine the impact of an educational program on the prevention of central venous catheter-related infections in a Brazilian Pediatric Intensive Care Unit. Patients and Methods: All patients admitted to the unit between February 2004 and May 2005 were included in the cohort study in a longitudinal assessment. An educational program was developed based on the Centers for Disease Control and Prevention recommendations for prevention of catheter-associated infections and was adapted to local conditions and resources after an initial observational phase. Incidence of catheter-associated infections was measured by means of on-site surveillance. Results: One hundred eighteen nosocomial infections occurred in 253 patients (46.6 infections per 100 admissions) and in 2,954 patient-days (39.9 infections per 1,000 patient-days). The incidence-density of catheter infections was 31.1 episodes per 1.000 venous central catheter-days before interventions, and 16.5 episodes per 1,000 venous central catheter-days afterwards (relative risk 0.53 [95% CI 0.28-1.01]). Corresponding rates for exit-site catheter infections were 8.0 and 2.5 episodes per 1,000 venous central catheter-days [0.32 (0.07-1.49)], and the rates for bloodstream infections were 23.1 and 13.9 episodes per 1,000 venous central catheter-days, before and after interventions [0.61 (0.32-1.14)]. Conclusion: A prevention strategy targeted at the insertion and maintenance of vascular access can decrease rates of vascular-access infections in pediatric intensive care unit.