New approaches to the treatment of influenza have been designed based on the highly conserved antigenicity of the M2 envelope protein among influenza A virus strains. The present study examined the anti-viral activities of an anti-M2 ectodomain (M2e) monoclonal antibody (clone rM2ss23), which binds to the M2 proteins of the influenza A virus strains A/ Aichi/2/68 (H3N2) (Aichi) and A/PJR/8/34 (H1N1) (PR8). The results showed that rM2ss23 bound to both Aichi and PR8 M2 proteins expressed on the cell surface. While the antibody did not prevent virus entry into cells, it significantly inhibited plaque formation by the Aichi strain in a dose-dependent manner when infected cells were cultured in the presence of the antibody. By contrast, the growth of PR8 (H1N1) was not affected by the antibody. A reverse genetics approach revealed that the inhibitory effect of rM2ss23 on the Aichi virus was abolished by replacing the genes encoding the HA and/or M proteins with those of the PR8 strain. These results suggest that rM2ss23 prevents virus release from infected cells and further suggest that the mechanisms underlying the virus budding mediates by HA and M2 proteins might differ between the Aichi and PR8 strains.