Hedgehog acyltransferase (Hhat) is an N-palmitoyltransferase that acylates Sonic hedgehog (Shh), which is critical for signaling through Shh. RU-SKI 43 is an inhibitor of Hhat (IC50 = 0.85 μM). It blocks palmitoylation of Shh without affecting palmitoylation of H-Ras or Fyn, myristoylation of c-Src, or acylation of Wnt3a. RU-SKI 43 is cell-permeable and inhibits both autocrine and paracrine Shh-induced activation of Gli-mediated transcription. In pancreatic cancer cells, RU-SKI 43 reduces both Gli1 activation and proliferation.