BACKGROUND/AIMS: The negative effects on the eradication success of Helicobacter pylori infection after previous exposure to macrolides, including clarithromycin on clarithromycin-based first-line therapy have been demonstrated. However, whether this is true for metronidazole-based second-line quadruple therapy remains unclear. We investigated the relationship between past administration of metronidazole and the failure of metronidazole-based second-line quadruple therapy in patients with H. pylori infection. METHODS: Patients over 20 years of age who were diagnosed with H. pylori infection between January 1998 and March 2016 were enrolled in this study. The relationship between the clinical parameters and the results of a C13-urea breath test after metronidazole-based second-line quadruple therapy was analyzed in patients for whom clarithromycin-based triple therapy failed to eradicate H. pylori. RESULTS: The H. pylori eradication failure rate was significantly higher in patients with a history of metronidazole use than in patients without a history of metronidazole use (p = 0.011). Multivariable analysis showed that the odds ratio of previous metronidazole use for eradication failure was 3.468 (95% confidence interval, 1.391 to 8.649; p = 0.008). In the subgroup analysis of patients with a history of metronidazole use, the duration of metronidazole use and interval between its use and eradication therapy did not significantly affect H. pylori eradication failure. CONCLUSION: Previous exposure to metronidazole was a significant risk factor for treatment failure of metronidazole-based second-line quadruple therapy; therefore, this should be considered when establishing a treatment strategy for patients with H. pylori infection.