Abstract A vaccine for Kaposi's sarcoma-associated herpesvirus (KSHV) is not currently available. To obtain the fundamental data in animals for vaccine development, KSHV particles were immunized to Balb/c mice through intraperitoneal and intranasal routes in the present study. Intranasal immunization with KSHV induced IgA to KSHV in not only serum, but also nasal wash fluid and saliva. A neutralization assay using recombinant KSHV that expressed green fluorescent protein revealed that nasal wash fluid and saliva from the KSHV-immunized mice neutralized KSHV infection to human embryonic kidney 293 cells in vitro in a dose-dependent manner to KSHV copies immunized. The serum and nasal wash fluid of KSHV-encoded K8.1 protein-immunized mice neutralized KSHV infection to 293 cells in vitro. These data suggest a possibility of mucosal vaccine for prophylaxis of KSHV infection.