Purpose: The aim of study was to estimate the hand hygiene (HH) compliance of healthcare workers (HCWs) in a children's hospital. Methods: This study was conducted in a hospital which is a tertiary and educational children's hospital with 313 beds and 533 HCWs. Data were collected by direct observation methods from November 1, 2010 to December 31, 2010. Results: A total of 2,999 opportunities for HH were observed, and the overall HH rate was 95.3%. HH rate of the registered nurse, physicians and transferer was 97.7%, 89.2%, and 72.1%, respectively (P<0.001). Among physicians, HH rate of the fellows, professors, residents and interns was 97.5%, 93.9%, 89.7%, and 80.9%, respectively (P<0.001). HH rate in the emergency room, operation room, outpatient department (OPD), and the intensive care unit (ICU) was 97.2%, 97.2%, 95.4%, and 92.5%, respectively (P<0.001). Hand rubbing was the most frequently used (81.1%), and hand washing was frequently used in the case of 'after body fluids exposure risk' (37.7%) and 'after touching patient surroundings' (28.5%). HH methods were not statistically different from each departments (P =0.083), however, they were significantly different according to the World Health Organization (WHO) 5 Moments (P<0.001). Distributions in WHO 5 Moments by the job titles were significantly different (P<0.001). The odds ratio of physicians, ICU and OPD was 0.353 (95% CI, 0.241-0.519), 0.291 (95% CI, 0.174-0.487), and 0.484 (95% CI, 0.281-0.834), respectively. Conclusions: Compliance of HH was different by the job titles and departments. Effective custom-tailored HH programs for each job title and department need to be developed.