PURPOSE: Accumulating evidence suggests that cancer-associated stromal fibroblasts (CAF) contribute to tumor growth by actively communicating with cancer cells. Our aim is to identify signaling pathways involved in tumor-stromal cell interactions in human pancreatic cancer. EXPERIMENTAL DESIGN: We established primary fibroblast cultures from human pancreatic adenocarcinomas and nonneoplastic pancreas tissues. To identify differentially expressed genes in CAFs, we did gene expression profiling of human pancreatic CAFs and nonneoplastic pancreatic fibroblasts. RESULTS: The Hedgehog receptor Smoothened (SMO) was upregulated in CAFs relative to control fibroblasts. CAFs expressing SMO could transduce the Sonic hedgehog signal to activate Gli1 expression, and small interfering RNA knockdown of SMO blocked the induction of Gli1 in these cells. Stromal fibroblasts of human primary pancreatic adenocarcinomas overexpressed Smo compared with normal pancreatic fibroblasts. CONCLUSIONS: These findings implicate overexpression of Smo as a mechanism for the activation of Hedgehog signaling in human pancreatic CAFs and suggest that stromal cells may be a therapeutic target for Smo antagonists in pancreatic cancer.