Because healthcare workers played an important role in the battle against novel pandemic (H1N1) 2009 influenza, a clinical study was conducted to examine the immunogenicity and safety of a single dose of a China-made monovalent, split-virus, pandemic (H1N1) 2009 influenza vaccine in this special high-risk population. Healthcare workers in the First Affiliated Hospital of Guangzhou Medical College who received the pandemic (H1N1) 2009 influenza vaccine were prospectively enrolled. Antibody titers were measured at enrollment and 14 days later using hemagglutination-inhibition (HI) and microneutralization assays. Adverse events were recorded within 7 days and 6 months after vaccination. Double sera were provided by 51 of 65 enrolled subjects. Postvaccination titers of 1:40 or more on HI assay were observed in 96% of recipients. Seroconversion or a significant increase in titer on HI assay occurred in 59% of subjects. The factor increase in GMTs was 4.3. The majority of complaints were mild to moderate in intensity. Although more than half of healthcare workers seemed immune to the pandemic (H1N1) influenza A virus before vaccination, most of the subjects still showed a fast, robust immune response to a single 15-μg dose of a monovalent, split-virus unadjuvanted pandemic H1N1 2009 influenza vaccine.