Olfactomedin 4 (OLFM4) is a glycoprotein that has been found to be up-regulated in inflammatory bowel diseases and Helicobacter pylori infected patients. However, its role in biological processes such as inflammation or other immune response is not known. In this study, we generated OLFM4 KO mice to investigate potential role(s) of OLFM4 in gastric mucosal responses to H. pylori infection. H. pylori colonization in the gastric mucosa of OLFM4 KO mice was significantly lower compared with WT littermates. The reduced bacterial load was associated with enhanced infiltration of inflammatory cells in gastric mucosa. Production and expression of proinflammatory cytokines/chemokines such as IL-1β, IL-5, IL-12 p70, and MIP-1α was increased in OLFM4 KO mice compared with infected controls. Furthermore, we found that OLFM4 is a target gene of NF--κB pathway and has a negative feedback effect on NF-κB activation induced by H. pylori infection through a direct association with nucleotide oligomerization domain-1 (NOD1) and -2 (NOD2). Together these observations indicate that OLFM4 exerts considerable influence on the host defense against H. pylori infection acting through NOD1 and NOD2 mediated NF-κB activation and subsequent cytokines and chemokines production, which in turn inhibit host immune response and contribute to persistence of H. pylori colonization.