Natural infection-induced humoral immunity to M2 protein of influenza A viruses in humans is not fully understood at present. Existing evidence suggests that anti-M2 antibody responses following influenza A virus infection are weak and/or transient. We show that seroprevalence of anti-M2 antibodies increased with age in 317 sera from healthy individuals in the U.S. in 2007-08. Infection with 2009 pandemic H1N1 influenza A virus [A(H1N1)pdm09] elicited a recall serum antibody response to M2 protein of the A(H1N1)pdm09 virus in 47% of the affected 118 individuals tested. Anti-M2 antibody responses were more robust among individuals with preexisting antibodies to M2 protein. Moreover, the antibodies induced as a result of infection with A(H1N1)pdm09 virus were cross-reactive with M2 protein of seasonal influenza A viruses. These results emphasize the need to further investigate the possible roles of anti-M2 antibodies in human influenza A virus infection.