Activation of the hedgehog (HH) pathway plays a critical role in the development and continued growth of pancreatic adenocarcinoma (PAC). Cyclopamine, a HH pathway inhibitor, has been shown to suppress PAC cell proliferation in vitro and in vivo. However, the molecular basis of response to cyclopamine has not been fully elucidated nor have genes that predict sensitivity to this compound been identified. To better understand these features of HH pathway inhibition, we evaluated the biological and molecular effects of cyclopamine in vitro. The viability of 9 human PAC cell lines following cyclopamine exposure was determined using MTS assay. Proliferation and induction of apoptosis in treated cells were examined by bromo-deoxyuridine incorporation, caspase activation, and mitochondrial membrane potential. Gene expression before and after cyclopamine treatment was determined using Taqman real-time quantitative polymerase chain reaction (RTQ-PCR) and Taqman low-density array (TLDA). Among the cell lines examined, cyclopamine IC50 values ranged from 8.79 to >30 µM. Response to cyclopamine included reduced cell proliferation and induction of apoptosis with and without mitochondrial membrane depolarization. Regression analysis revealed that GLI3 expression significantly correlated with cyclopamine resistance (r = 0.80; p = 0.0102). Knockdown of GLI3 using siRNAs increased sensitivity to cyclopamine. In addition, GLI3 siRNAs decreased PAC cell viability and reduced expression of genes involved in HH signaling (Patched 1 and GLI1) and cell proliferation, similar to cyclopamine. These effects were not observed in PAC cells with undetectable GLI3 expression. These data suggest that Gli3 mediates cell survival and sensitivity to cyclopamine in pancreatic cancer.