After the emergence of influenza A viruses in the human population, the number of N-glycosylation sites (NGS) in the globular head region of hemagglutinin (HA) has increased continuously for several decades. It has been speculated that the addition of NGS to the globular head region of HA has conferred selective advantages to the virus by preventing the binding of antibodies (Ab) to antigenic sites (AS). Here the effect of N-glycosylation on the binding of Ab to AS in human influenza A virus subtype H3N2 (A/H3N2) was examined by inferring natural selection at AS and other sites (NAS) that are closely and distantly located from the NGS in the three-dimensional structure of HA through a comparison of the rates of synonymous (d(S)) and nonsynonymous (d(N)) substitutions. When positions 63, 122, 126, 133, 144, and 246 in the globular head region of HA were non-NGS, d(N)/d(S) was > 1 and positive selection was detected at the AS closely located to these positions. However, the d(N)/d(S) value decreased and the evidence of positive selection disappeared when the above positions became NGS. In contrast, d(N)/d(S) at the AS distantly located from the above positions and at the NAS of any location were generally < 1, and did not decrease when the above positions changed from non-NGS to NGS. These results suggest that the attachment of N-glycans to the NGS in the globular head region of HA prevented the binding of Ab to AS in the evolutionary history of human A/H3N2 virus.