Helicobacter pylori (H. pylori) is a bacterial pathogen in the stomach, causing gastritis, gastric ulcer, duodenal ulcer and even gastric cancer. The triple therapy containing one bismuth-containing compound or a proton-pump inhibitor with two antibiotics was the cornerstone of the treatment of H. pylori infections. However the drug resistance of Helicobacter pylori is more and more common, which leads to the continued decline in the radical cure rate. The purpose of this study was to investigate the mechanism of metronidazole resistance of H. pylori through transcriptomics and biochemical characterizations. In this study, a 128-time-higher metronidazole-resistant H. pylori strain compared to the sensitive strain was domesticated, and 374 significantly differential genes were identified by transcriptomic sequencing as compared to the metronidazole-sensitive strain. Through GO and KEGG enrichment analysis, antibiotic-resistance pathways were found to be mainly involved in redox, biofilm formation and ABC transportation, and the results were verified by qRT-PCR. The subsequent biochemical analysis found that the urease activity of the drug-resistant strain decreased, and whereas the capabilities of bacterial energy production, membrane production and diffusion ability increased. The work here will drop hints for the mechanisms of antibiotic-resistance of H. pylori and provide promising biomarkers for the further development of new-kind drugs to treat metronidazole-resistant H. pylori.