Background: A pandemic H5N1 influenza outbreak would be facilitated by an absence of immunity to the avian-derived virus in the human population. Although this condition is likely in regard to hemagglutinin-mediated immunity, the neuraminidase (NA) of H5N1 viruses (avN1) and of endemic human H1N1 viruses (huN1) are classified in the same serotype. We hypothesized that an immune response to huN1 could mediate cross-protection against H5N1 influenza virus infection. Methods and Findings: Mice were immunized against the NA of a contemporary human H1N1 strain by DNA vaccination. They were challenged with recombinant A/Puerto Rico/8/34 (PR8) viruses bearing huN1 (PR8-huN1) or avN1 (PR8-avN1) or with H5N1 virus A/Vietnam/1203/04. Additional naïve mice were injected with sera from vaccinated mice prior to H5N1 challenge. Also, serum specimens from humans were analyzed for reactivity with avN1. Immunization elicited a serum IgG response to huN1 and robust protection against the homologous challenge virus. Immunized mice were partially protected from lethal challenge with H5N1 virus or recombinant PR8-avN1. Sera transferred from immunized mice to naïve animals conferred similar protection against H5N1 mortality. Analysis of human sera showed that antibodies able to inhibit the sialidase activity of avN1 exist in some individuals. Conclusions: These data reveal that humoral immunity elicited by huN1 can partially protect against H5N1 infection in a mammalian host. Our results suggest that a portion of the human population could have some degree of resistance to H5N1 influenza, with the possibility that this could be induced or enhanced through immunization with seasonal influenza vaccines.