Swine influenza A virus (swIAV) is an important pathogen with regard to both the swine industry and public health. The pandemic A(H1N1) 2009 outbreak was caused by the swine-origin pandemic A(H1N1) 2009 [A(H1N1)pdm09] virus. Several reports have shown that several amino acid substitutions in the hemagglutinin (HA) antigenic sites can alter HA antigenicity. However, the impact of the amino acid deletion at position 155 on HA antigenicity remains unknown. In this study, we have isolated 11 samples of swIAVs from seven pig farms in Japan and found an amino acid deletion at position 155 of the HA region in one of the isolates of the H1N2 subtype. To examine the impact of this amino acid deletion on viral replication and HA antigenicity, we generated recombinant influenza A viruses possessing the H1 HA gene encoding either an artificial insertion or deletion of glycine at position 155. The growth kinetics of these recombinant viruses in two different cell lines demonstrated that the effect of amino acid deletion at position 155 of H1 HA on viral replication is limited. In contrast, microneutralization assay-based neutralization titers revealed that amino acid deletion significantly altered HA antigenicity. These results demonstrate that a naturally occurring amino acid deletion at position 155 in an H1 HA antigenic site can markedly alter HA antigenicity with only a limited impact on replication in vitro, highlighting the need to monitor such variants in swine populations and to assess their zoonotic potential.