Background: Recently, the number of Helicobacter pylori isolates showing antibiotic resistance has been increasing. Rifabutin (RFB) is one of the possible candidates for H. pylori eradication. In the present study, the RFB minimum inhibitory concentrations (MICs) and the resistance-determining genes to RFB (rpoB) were examined to clarify the relationship between drug MICs, rpoB mutations, and past history of rifampicin (RFP) treatment. Methods: The MICs of RFB and rpoB mutations were examined for 48 strains with failure of H. pylori eradication in the University Hospital and 46 isolated from patients at a specialized hospital for chronic respiratory diseases without past H. pylori eradication. Past RFP treatment was also examined. Results: Eight of 94 strains showed high RFB MICs and 6 of the 8 strains showed rpoB point mutations. Although no strains showed high RFB MICs among 48 strains from the patients in the University Hospital, all 7 strains isolated from patients with past RFP treatment showed high RFB MICs (≥0.12 mg/l). Conclusion: Although RFB might be a potential candidate component of a new H. pylori eradication regimen following the first- or second-line failure, it should be used after examining a past history of RFP treatment.