Passive immunization with human H5 antisera or H5-specific monoclonal antibodies (MAbs) has potential as an effective treatment for acute H5N1 influenza virus infection, but its efficacy against antigenically diverse H5N1 viruses is unconfirmed.Cross-protection against antigenically diverse H5N1 strains with H5-specific MAbs, generated by successive immunization of antigenically distinct strains, was evaluated in mice.A panel of 52 broadly cross-reactive H5 specific MAbs were generated and characterized. One of these MAbs, 13D4, has been demonstrated to protect mice against lethal challenge by 4 H5N1 strains representing the current major genetic populations, clades 1, 2.1, 2.2, and 2.3, even at a stage of infection when H5N1 virus has disseminated beyond the pulmonary system. Complete neutralization of virus in lung tissue of infected animals was observed 24 h after treatment with 13D4. Mapping of this MAb with escape mutants showed it to bind to 2 conserved, possibly critical, sites of H5N1 hemagglutinin, 152 and 182.Generation of broadly cross-protective MAbs against H5N1 influenza virus may be optimized by selecting MAbs that target conserved sites in hemagglutinin. H5 MAbs such as 13D4 may prove to have therapeutic value in controlling infection due to current and future H5N1 variants.