We investigated the beta-lactam resistance mechanism(s) of an in vitro-selected amoxicillin-resistant Helicobacter pylori strain (AmoxR). Our results demonstrated that resistance is due to a combination of amino acid substitutions in penicillin binding protein I (PBP1), HopB, and HopC identified in AmoxR, resulting in decreased affinity of PBP1 for amoxicillin and decreased accumulation of penicillin.