Wnt signalling stimulated by binding of R-spondin (Rspo) to Lgr-family members is crucial for gastrointestinal stem cell renewal. Infection of the stomach with Helicobacter pylori stimulates increased secretion of Rspo by myofibroblasts, leading to an increase in proliferation of Wnt-responsive Axin2⁺Lgr5⁻ stem cells in the isthmus of the gastric gland and finally gastric gland hyperplasia. Basal Lgr5⁺ cells are also exposed to Rspo3, but their response remains unclear. Here, we demonstrate that—in contrast to its known mitogenic activity—Rspo3 induces differentiation of basal Lgr5⁺ cells into secretory cells that express and secrete antimicrobial factors, such as intelectin-1, into the lumen. The depletion of Lgr5⁺ cells or the knockout of Rspo3 in myofibroblasts leads to hypercolonization of the gastric glands with H. pylori, including the stem cell compartment. By contrast, systemic administration or overexpression of Rspo3 in the stroma clears H. pylori from the gastric glands. Thus, the Rspo3–Lgr5 axis simultaneously regulates both antimicrobial defence and mucosal regeneration.