Background: Most Japanese adults have been vaccinated twice in childhood with the Sabin oral polio vaccine as part of routine immunization schedules. Booster vaccination is recommended for Japanese travelers to polio-endemic or high-risk countries. This study assessed the immunogenicity of a two-dose booster regimen of conventional inactivated polio vaccine (cIPV) in Japanese adults.Methods: Healthy Japanese adults aged 20 years or older received two doses of standalone cIPV derived from virulent strains (Imovax Polio®). Serum samples were obtained before the booster vaccination and 4–6 weeks after each vaccination. Immunogenicity was evaluated by measuring serum neutralization titers against type 1, 2, and 3 poliovirus strains using a microneutralization assay. Results: The subjects were 61 healthy Japanese adults (26 men and 35 women) with mean ± standard deviation age of 35.8 ± 8.0 years. The seropositivity rates (i.e., percentage of subjects with anti-polio antibody titers ≥1:8 ) before booster vaccination were 88.5%, 95.1%, and 52.5% for Sabin strains (type 1, 2, and 3, respectively); 72.1%, 93.4%, and 31.1% for virulent poliovirus strains (type1: Mahoney strain; type 2: MEF-1 strain; and type 3: Saukett strain, respectively); and 93.4%, 93.4%, 93.4% and 88.5% for type 2 vaccine-derived poliovirus strains (SV3128, SV3130, 11196, and 11198, respectively). After one dose of cIPV, all seropositivity rates increased to 98.4%–100.0%. After two doses of cIPV, seropositivity rates reached 100% for all strains. cIPV was well tolerated, with no safety concerns. Conclusion: Booster vaccination with standalone cIPV induced a robust immune response in Japanese adults. Trial registration: UMIN, UMIN000013551. Registered 28 March 2014 - Retrospectively registered, https://upload.umin.ac.jp/cgi-bin/ctr/ctr_view_reg.cgi?recptno=R000015830