Acidic media trigger cytoplasmic urease activity of the unique human gastric pathogen Helicobacter pylori. Deletion ofureI prevents this activation of cytoplasmic urease that is essential for bacterial acid resistance. UreI is an inner membrane protein with six transmembrane segments as shown by in vitro transcription/translation and membrane separation. Expression of UreI in Xenopus oocytes results in acid-stimulated urea uptake, with a pH profile similar to activation of cytoplasmic urease. Mutation of periplasmic histidine 123 abolishes stimulation. UreI-mediated transport is urea specific, passive, nonsaturable, nonelectrogenic, and temperature independent. UreI functions as a H⁺-gated urea channel regulating cytoplasmic urease that is essential for gastric survival and colonization.