Infection of human gastric body mucosa by the Gram-negative, microaerophilic bacterium Helicobacter pylori induces an inflammatory response and a transitory hypochlorhydria which progresses in ~2% of patients to atrophic gastritis, dysplasia, and gastric adenocarcinoma. We have previously shown that H. pylori infection of cultured gastric epithelial cells (AGS) represses the activity of transfected subunit (HK) promoter of H,K-ATPase, the parietal cell enzyme mediating acid secretion. However, the mechanistic details of H. pylori-mediated repression of HK and ensuing hypochlorhydria are unknown. H. pylori is known to up-regulate the transcription factor NF-B through the ERK 1/2 MAPK pathway. We identified NF-B binding regions in HK promoter and found that H. pylori inoculation of AGS cells increased NF-B p50 binding to transfected HK promoter and repressed its transcriptional activity. Immunoblot and DNA-protein interaction studies showed that although active phosphorylated NF-B p65 is present in H. pylori-infected AGS cells, an NF-B p50/p65 heterodimeric complex fails to bind to HK promoter. Point mutations at -159 and -161 bp in HK promoter NF-B binding sequence prevented binding of NF-B p50 and prevented H. pylori repression of point-mutated HK promoter activity in transfected AGS cells. siRNA-mediated knockdown of NF-B p50 in H. pylori-infected AGS cells also abrogated H. pylori-induced HK repression, whereas NF-B p65 knockdown did not. We conclude that H. pylori inhibits HK gene expression by ERK 1/2-mediated NF-B p50 homodimer binding to HK promoter. This study identifies a novel pathogen-dependent mechanism of H,K-ATPase inhibition and contributes to understanding of H. pyloripathophysiology.