The differential antibody response measured by the commonly used hemagglutination inhibition (HI) and microneutralization (MN) assays in patients with natural infection and vaccination has not been fully assessed. HI and conventional MN (CMN) assays were performed on sera from 651 patients with natural infection by pandemic H1N1 2009 influenza virus and on sera from 567 recipients of the corresponding vaccine. Surprisingly, the overall seroprotection rates determined by CMN and HI assays in vaccine recipients were only 44.8 and 35.1%, respectively. Antibody titers measured by the CMN assay was significantly higher than that obtained by HI assay in vaccine recipients aged ≥50 years, but these titers were not significantly different among younger vaccine recipients. In contrast, the HI titer was greater than the CMN titer for the age group from 16 to 29 years but was not significantly different in other age groups for natural infection. Lower antibody levels were found in both naturally infected patients and immunized recipients in the older than in the younger age groups, but naturally infected patients exhibited higher HI and CMN titers than did the corresponding vaccine recipients. In addition, we developed a rapid fluorescent focus microneutralization (FFMN) assay to test sera from naturally infected patients. The FFMN assay has a better correlation with CMN than with HI (ρ = 0.810 versus 0.684), which is expected of neutralizing antibody mainly targeted toward the inhibition of viral entry into cells. The higher antibody level elicited by natural infection than by vaccination may be related to differences between antigen presentation by the intramuscular route of vaccination and mucosal viral replication in mucosal cells of the respiratory tract.