The Shh pathway has been implicated in gastric carcinogenesis, and inhibition of this pathway has been shown to inhibit tumour growth in gastric cell lines. Assessing the in vivo efficacy of Shh pathway antagonists in blocking Shh signaling in the stomach is important for clinical trial design, but has not been previously investigated. We investigated the in vivo efficacy of a Shh antagonist, cyclopamine, in correlation to the secondary effects induced by this treatment on gastrin levels and acid secretion. Gastrin has been shown to induce Shh production, processing and activity, which is believed to be mediated by acid secretion. We tested this hypothesis and showed that hypergastrinaemia induces Shh production in vivo, and confirmed that this effect on Shh is mediated by acid secretion. We showed that cyclopamine treatment induces both hypergastrinaemia and Shh, and does not inhibit Gli-1. Inhibition of the effect of hypergastrinaemia on the Shh pathway, in cyclopamine-treated mice, was demonstrated by use of lansoprazole which concomitantly inhibited Gli-1, and did not increase Shh production. Therefore, this evidence suggests that hypergastrinaemia, via increased acid secretion, may increase expression of Shh and that Shh antagonists may require concomitant acid inhibition to successfully inhibit a pathway known to be up-regulated in gastric carcinogenesis.