AIM: To determine changes in the antibiotic resistance of Helicobacter pylori(H. pylori) in southern Taiwan after failure of first-line standard triple therapy.METHODS: We analyzed 137 H. pylori-infected isolates from patients who experienced eradication failure after standard first-line triple therapy from January2010 to December 2014. The H. pylori strains were tested for susceptibility to amoxicillin, clarithromycin,levofloxacin, metronidazole and tetracycline using the E-test method. The minimal inhibitory concentration(MIC) was determined by the agar dilution test.MIC values of ≥ 0.5, ≥ 1, ≥ 1, ≥ 4 and ≥ 8 mg/L were considered to be the resistance breakpoints for amoxicillin, clarithromycin, levofloxacin, tetracycline and metronidazole, respectively.RESULTS: A high resistance rate was found for clarithromycin(65%-75%) and metronidazole(30%-40%)among patients who failed first-line standard therapy.The resistance levels to amoxicillin and tetracycline remained very low; however, levofloxacin resistance was as high as 37.5% in 2010 but did not increase any further during the past 5 years. The rates of resistance to these antibiotics did not show a statistically significant upward or downward trend.CONCLUSION: Antibiotic resistance of H. pylori remains a problem for the effective eradication of this pathogen and its associated diseases in Taiwan. High clarithromycin resistance indicated that this antibiotic should not be prescribed as a second-line H. pylori eradication therapy. Moreover, levofloxacin-based second-line therapy should be used cautiously, and the local resistance rates should be carefully monitored.