Bartonella henselae, a small fastidious Gram-negative bacillus, is the causative agent of cat-scratch disease (CSD). Because of difficulty in isolating the organism, there has been no report on its antibiotic susceptibility in Japan. We determined the minimal inhibitory concentrations (MICs) of eight antimicrobial agents against 32 isolates of B. henselae (31 from cats and one from a human in Japan) by the Etest method. MICs of all 32 isolates were <0.016 μg/ml for minocycline and ranged from ≤0.016 to 0.064 μg/ml for erythromycin, clarithromycin, azithromycin, ceftriaxone, and amoxicillin. MICs ranges of ciprofloxacin and gentamicin were from 0.064 to 0.25 μg/ml and from 0.5 to 3 μg/ml, respectively. All isolated strains showed high susceptibility to minocycline and macrolides antibiotics, which are currently used in the primary treatment of CSD in Japan. Although in vitro result of B. henselae susceptibility testing may not necessarily correlate with clinical response, these data are relevant in the choice of drugs for CSD treatment.