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• HV004 strain showed higher proliferation efficiency and pathogenicity. • The inactivated HTNV vaccine was administered safely in immunized BALB/C mice. • The vaccine induced sufficient neutralizing antibodies and cellular immune response. Hantaan virus (HTNV, Orthohantavirus hantanensae species, Hantaviridae family) is the main etiological agent responsible for hemorrhagic fever with renal syndrome (HFRS). The novel HTNV may pose a potential danger to the control and prevention of HFRS in China, which highlights the importance of vaccine development in public health management. In previous studies, our laboratory discovered and successfully isolated a new HTNV strain, HV004 strain, from Apodemus agrarius captured in an epidemic area in Hubei, China. An initial biological and pathogenicity characterization of HTNV 76–118 (standard train), HV114 strain (a clinical isolate from Hubei province in 1986), and the novel isolate HV004 strain from the epidemic areas of Hubei province were performed in susceptible cells and in vivo. An experimental HV004 strain inactivated vaccine was prepared, and its corresponding immunogenicity was analyzed in BALB/c mice. HV004 strain had a similar but higher pathogenicity than HTNV 76–118 and HV114 in suckling mice. A subcutaneous vaccination (s.c.) with the inactivated HTNV vaccine adjuvanted with aluminum, followed by a challenge intraperitoneally with 106 FFU/ml HTNV, afforded full protection against an HTNV challenge. All immunized mice in every group elicited serum neutralizing antibodies with increasing dosages, which may protect mice from HTNV infection. A dose-dependent stimulation index of splenocytes was also observed in immunized mice. The percentage of IFN-γ-producing CD3+CD8+ T cells was significantly higher in the spleens of immunized mice than in those of control mice. These findings suggest that the inactivated HTNV vaccine may stimulate mice to produce high levels of antibodies with neutralization activity and elicit specific anti-HTNV humoral and cellular immune responses in BALB/c mice against the prevalent strain of HTNV in south central China. [ABSTRACT FROM AUTHOR]