Hantaan virus (HTNV), a member of the genus Hantavirus, family Bunyaviridae, is an etiologic agent causing a serious human disease, hemorrhagic fever with renal syndrome (HFRS), with a mortality as high as 15% and is also a potential bioterrorism agent. It is urgently needed to develop anti-HTNV-neutralizing monoclonal antibodies (MAbs) for treatment and prevention of HTNV infection. In the present study, 18 murine MAbs directed against HTNV strain Chen were generated and characterized. Among these MAbs, 13 were directed against viral nucleocapsid protein (NP), four recognized the viral envelope glycoprotein G2 and one reacted with both NP and G2. Only those MAbs that recognize the epitopes on G2 were positive in hemagglutination inhibition (HI) test and had in vitro virus-neutralizing activity and in vivo protective activity against HTNV infection of susceptible mice. Since all the mice were protected by administration of the virus-neutralizing MAbs one day before and two days after HTNV challenge, these neutralizing MAbs are potentially useful for pre- and post-exposure prophylaxis and for immunotherapy of HTNV infection. Phase II clinical trials of these neutralizing MAbs for emergent treatment of patients with HTNV infection in early stages of HRFS are carried out in endemic areas in China.