OBJECTIVE: The effectiveness of Helicobacter pylori therapies has declined with an increase in antibiotic resistance. To overcome this problem, the efficacy of tailored H. pylori eradication therapy based on antimicrobial susceptibility testing was compared with that of empirical second-line rescue regimens. MATERIAL AND METHODS: Patients who had persistent H. pylori infection after the first eradication were recommended to undergo culture for determining the minimal inhibitory concentration (MIC) via gastroscopy, which increased the cost by 300%. Fourteen-day esomeprazole, tripotassium dicitrate bismuthate, metronidazole and tetracycline (EBMT) therapy or esomeprazole, moxifloxacin and amoxicillin (MEA) therapy was performed according to the results of antibiotic susceptibility testing. In case of refusal to undergo culture, the participants were treated with either 14-day empirical EBMT or MEA regimen for second eradication after explaining the complexity, side effects and costs associated with each regimen. This trial was registered at ClinicalTrials.Gov (NCT 02349685). RESULTS: In the 219 patients included, the intention to treat (ITT) and per protocol (PP) eradication rates was 75.3% and 79.8% in the 14-day EBMT group (n = 89), 70.8% and 72.4% in the 14-day MEA group (n = 89) and 87.8% and 100.0% in the 14-day tailored therapy group (n = 41), respectively. Based on the PP analysis, the 14-day tailored therapy group showed a significantly higher eradication rate than the 14-day EBMT or MEA group (both p ≤ 0.001). CONCLUSIONS: Tailored therapy based on H. pylori culture and MIC test could be an option as a second-line eradication regimen in the presence of high level of antimicrobial resistance.