Helicobacter pylori cagA-positive strains are critically involved in the development of gastric cancer. Upon delivery into gastric epithelial cells via type IV secretion, the cagA-encoded CagA interacts with and thereby perturbs the pro-oncogenic phosphatase SHP2 and the polarity-regulating kinase PAR1b via the tyrosine-phosphorylated EPIYA-C/D segment and the CM sequence, respectively. Importantly, sequences spanning these binding regions exhibit variations among CagA proteins, which influence the pathobiological/oncogenic potential of individual CagA. Here we isolated an H. pylori strain (Hp_TH2099) naturally infecting the stomach of a housed macaque, indicating a zoonotic feature of H. pylori infection. Whole genome sequence analysis revealed that Hp_TH2099 belongs to the hpAsia2 cluster and possesses ABC-type Western CagA, which contains hitherto unreported variations in both EPIYA-C and CM sequences. The CM variations almost totally abolished PAR1b binding. Whereas pTyr + 5 variation in the EPIYA-C segment potentiated SHP2-binding affinity, pTyr-2 variation dampened CagA tyrosine phosphorylation and thus impeded CagA-SHP2 complex formation. As opposed to the H. pylori standard strain, infection of mouse ES cell-derived gastric organoids with Hp_TH2099 failed to elicit CagA-dependent epithelial destruction. Thus, the macaque-isolated H. pylori showed low virulence due to attenuated CagA activity through multiple substitutions in the sequences involved in binding with SHP2 and PAR1b.