[Objective] Benzathine benzylpenicillin (BPG) has been used as a standard treatment for syphilis worldwide with exception of Japan. BPG was nominated by the unapproved and off-label use committee as high medical need in 2017. The current study investigated the pharmacokinetics (PK) and safety of a single intramuscular (IM) injection of 2.4 million units (MU) of BPG in Japanese healthy adult participants. [Method] A total of 8 Japanese healthy participants were enrolled into the study to receive a single IM injection of 2.4 MU of BPG as a pre-filled syringe formulation. PK samples were collected up to 28 days after injection to measure the plasma concentrations of penicillin G (PCG). Non-compartment analysis was conducted to calculate the PK parameters of PCG. [Results・Discussion] Following a single IM injection of BPG, PCG was absorbed slowly from the injection site with a median time for Cmax (Tmax) of 48 hours post-dose. After the achievement of Cmax, PCG concentrations declined slowly in a monophasic fashion with mean terminal half-life of 188.8 hours. The geometric means of AUCinf and Cmax were 50770 ng.h/mL and 259.0 ng/mL, respectively. The median time (range) above the target efficacious concentration (18 ng/mL) for syphilis treatment was 561.0 hours (439-608 hours [18-25 days]). There was only 1 (12.5%) participant reported a non-serious AE of nasopharyngitis of mild intensity which was recovered and considered not related to study treatment. [Conclusion] A single IM injection of 2.4 MU of BPG reached and maintained the target efficacious PCG concentration over the necessary duration of 7 to 10 days for syphilis treatment. Along with the good safety profile in healthy Japanese participants and about 70 years of worldwide syphilis treatment experiences, BPG is considered a valid and essential treatment for Japanese syphilis patients.