Health care workers (HCWs) are at great risk of influenza infection and transmission. Vaccination for seasonal influenza is routinely recommended, but this strategy should be reconsidered in a pandemic situation. Between October 2009 and September 2010, a multicenter study was conducted to assess the long-term immunogenicity of the A/H1N1 2009 monovalent influenza vaccine among HCWs compared to non-health care workers (NHCWs). The influence of prior seasonal influenza vaccination was also assessed with respect to the immunogenicity of pandemic H1N1 influenza vaccine. Serum hemagglutinin inhibition titers were determined prevaccination and then at 1, 6, and 10 months after vaccination. Of the 360 enrolled HCW subjects, 289 participated in the study up to 10 months after H1N1 monovalent influenza vaccination, while 60 of 65 NHCW subjects were followed up. Seroprotection rates, seroconversion rates, and geometric mean titer (GMT) ratios fulfilled the European Union's licensure criteria for influenza A/California/7/2009 (H1N1) at 1 month after vaccination in both the HCWs and NHCWs, without any significant difference. At 6 months after vaccination, the seroprotection rate was more significantly lowered among the NHCWs than among the HCWs (P < 0.01). Overall, postvaccination (1, 6, and 10 months after vaccination) GMTs for A/California/7/2009 (H1N1) were significantly lower among the seasonal influenza vaccine recipients than among the nonrecipients (P < 0.05). In conclusion, HCWs should be encouraged to receive an annual influenza vaccination, considering the risk of repeated exposure. However, prior reception of seasonal influenza vaccine showed a negative influence on immunogenicity for the pandemic A/H1N1 2009 influenza vaccine.