Helicobacter pylori infection of the gastric body induces transient hypochlorhydria and contributes to mucosal progression toward gastric carcinoma. Acid secretion is mediated by parietal cell H,K-ATPase, in which the catalytic α-subunit (HKα) promoter activity in transfected gastric epithelial [gastric adenocarcinoma (AGS)] cells is repressed by H. pylori through NF-κB p50 homodimer binding to the promoter. IL-1β, an acid secretory inhibitor whose mucosal level is increased by H. pylori, upregulates HKα promoter activity in AGS cells. Because IL-1β also activates NF-κB signaling, we investigated disparate HKα regulation by H. pylori and IL-1β, testing the hypothesis that IL-1β-induced HKα promoter activation is mediated by the transcription factor Sp1. DNase I footprinting revealed Sp1 binding to the HKα promoter at −56 to −39 bp. IL-1β stimulated the activity of three HKα promoter constructs containing NF-κB and Sp1 sites transfected into AGS cells and also stimulated a construct containing only an Sp1 site. This stimulation was abrogated by mutating the HKα promoter Sp1 binding site. Gelshift assays showed that IL-1β increased Sp1 but not p50 binding to cognate HKα probes and that Sp1 also interacts with an HKα NF-κB site when bound to its cognate HKα cis-response element. H. pylori did not augment Sp1 binding to an HKα Sp1 probe, and small interfering RNA-mediated knockdown of Sp1 expression abrogated IL-1β-induced HKα promoter stimulation. We conclude that IL-1β upregulates HKα gene transcription by inducing Sp1 binding to HKα Sp1 and NF-κB sites and that the H. pylori perturbation of HKα gene expression is independent of Sp1-mediated basal HKα transcription.