Rodent-derived strains ofLactobacillus reuteridensely colonize the forestomach of mice and possess several genes whose predicted functions constitute adaptations towards an acidic environment. The objective of this study was to systematically determine which genes ofL. reuteri100-23 contribute to tolerance towards host gastric acid secretion. Genes predicted to be involved in acid resistance were inactivated, and their contribution to survival under acidic conditions was confirmed in model gastric juice. Fitness of five mutants that showed impairedin vitroacid resistance were then compared through competition experiments in ex-germ-free mice that were either treated with omeprazole, a proton-pump inhibitor that suppresses acid secretion in the stomach, or left untreated. This analysis revealed that the urease cluster was the predominant factor in mediating resistance to gastric acid production. Population levels of the mutant, which were substantially decreased in untreated mice, were almost completely restored through omeprazole, demonstrating that urease production inL. reuteriis mainly devoted to overcome gastric acid. The findings provide novel information on the mechanisms by whichL. reutericolonizes its gastric niche and demonstrate thatin silicogene predictions andin vitrotests have limitations for predicting the ecological functions of colonization factors in bacterial symbionts.