Background: Eradication rates for current H. pylori therapies have fallen in recent years, in line with the emergence of antibiotic resistant infections. The development of therapeutic alternatives to antibiotics, such as immunomodulatory therapy and vaccines, requires a more lucid understanding of host–pathogen interactions, including the relationships between the organism and the innate immune response. Pellino proteins are emerging as key regulators of immune signaling, including the Toll‐like receptor pathways known to be regulated by H. pylori. The aim of this study was to characterize the role of Pellino proteins in the innate immune response to H. pylori lipopolysaccharide. Materials and Methods: Gain‐of‐function and loss‐of‐function approaches were utilized to elucidate the role of individual Pellino proteins in the Toll‐like receptor 2‐mediated response to H. pylori LPS by monitoring NF‐ĸB activation and the induction of proinflammatory chemokines. Expression of Pellino family members was investigated in gastric epithelial cells and gastric tissue biopsy material. Results: Pellino1 and Pellino2 positively regulated Toll‐like receptor 2‐driven responses to H. pylori LPS, whereas Pellino3 exerted a negative modulatory role. Expression of Pellino1 was significantly higher than Pellino3 in gastric epithelial cells and gastric tissue. Furthermore, Pellino1 expression was further augmented in gastric epithelial cells in response to infection with H. pylori or stimulation with H. pylori LPS. Conclusions: The combination of low Pellino3 levels together with high and inducible Pellino1 expression may be an important determinant of the degree of inflammation triggered upon Toll‐like receptor 2 engagement by H. pylori and/or its components, contributing to H. pylori‐associated pathogenesis by directing the incoming signal toward an NF‐kB‐mediated proinflammatory response.