Background  Amoxicillin‐resistant Helicobacter pylori with minimal inhibitory concentration (MIC) ≥ 256 mg L−1 was isolated from a gastritis patient. The aims were to investigate the mechanism of high‐level amoxicillin resistance in H. pylori. Materials and methods  The β‐lactamase production was determined by means of nitrocefin sticks and the presence of gene encoding the β‐lactam antibiotic resistance enzyme TEM β‐lactamase was analysed by polymerase chain reaction (PCR), sequencing and dot‐blot hybridization. Sequencing analysis of pbp1A gene was performed and amoxicillin‐susceptible isolate was transformed with pbp1A PCR products from the resistant isolate. The expression of hefC efflux system was analysed using real‐time quantitative PCR. Results  Activity of β‐lactamase was detected. Sequence analysis showed that the PCR product derived from H. pylori 3778 was identical to the blaTEM‐1 (GenBank accession EU726527). Dot‐blot hybridization confirmed the presence of β‐lactamase gene blaTEM‐1. By transformation of PCR product of mutated pbp1A gene from H. pylori 3778 into amoxicillin‐susceptible strain showed that substitutions in Thr556→Ser, Lys648→Gln, Arg649→Lys and Arg656→Pro contribute to low‐level amoxicillin resistance. The MIC of amoxicillin for the transformants was 0·75 mg L−1. Over‐expression of hefC was not found. Conclusions  High‐level amoxicillin resistance is associated with β‐lactamase production in H. pylori. Low‐level amoxicillin resistance is linked to a point mutation on pbp1A. Because H. pylori can exchange DNA through natural transformation, spreading of blaTEM‐1 amoxicillin resistance gene among H. pylori is a potential threat when treating H. pylori infection.