Helicobacter pylori uses natural competence and homologous recombination to adapt to the dynamic environment of the stomach mucosa and maintain chronic colonization. Although H. pylori competence is constitutive, its rate of transformation is variable, and little is known about factors that influence it. To examine this, we first determined the transformation efficiency of H. pylori strains under low O2 (5% O2, 7.6% CO2, 7.6% H2) and high O2 (15% O2, 2.9% CO2, 2.9% H2) conditions using DNA containing an antibiotic resistance marker. H. pylori transformation efficiency was 6- to 32-fold greater under high O2 tension, which was robust across different H. pylori strains, genetic loci, and bacterial growth phases. Since changing the O2 concentration for these initial experiments also changed the concentrations of CO2 and H2, transformations were repeated under conditions where O2, CO2, and H2 were each varied individually. The results showed that the increase in transformation efficiency under high O2 was largely due to a decrease in CO2. An increase in pH similar to that caused by low CO2 was also sufficient to increase transformation efficiency. These results have implications for the physiology of H. pylori in the gastric environment, and they provide optimized conditions for the laboratory construction of H. pylori mutants using natural transformation.