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BACKGROUND: Concerns have been raised about how the transmission of emerging infectious diseases from patients to healthcare workers (HCWs) and vice versa could be recognized and prevented in a timely manner. An effective strategy to block transmission of pandemic H1N1 (2009) influenza in HCWs is important. METHODOLOGY/PRINCIPAL FINDINGS: An infection control program was implemented to survey and prevent nosocomial outbreaks of H1N1 (2009) influenza at a 2,600-bed, tertiary-care academic hospital. In total, 4,963 employees at Kaohsiung Chang Gung Memorial Hospital recorded their temperature and received online education on control practices for influenza infections. Administration records provided vaccination records and occupational characteristics of all HCWs. Early recognition of a pandemic H1N1 (2009) influenza case was followed by a semi-structured questionnaire to analyze possible routes of patient contact, household contact, or unspecified contact. Surveillance spanned August 1, 2009 to January 31, 2010; 51 HCWs were confirmed to have novel H1N1 (2009) influenza by quantitative real-time reverse transcription polymerase chain reaction. Prevalence of patient contact, household contact, or unspecified contact infection was 13.7% (7/51), 13.7% (7/51), and 72.5% (37/51), respectively. The prevalence of the novel H1N1 infection was significantly lower among vaccinated HCWs than among unvaccinated HCWs (p