Purpose: To investigate the effect and possible mechanisms of resveratrol on pancreatic cancer cells in vitro. Methods: After being treated with resveratrol, cell viability, cell cycle phase distribution and apoptosis rate of pancreatic cancer cells were measured by CCK-8 assay and flow cytometer, respectively. The effects of resveratrol on the Hedgehog pathway were studied by real-time RT-PCR and Western blotting. By interfering Gli1 expression in PANC-1 cells and overexpressing Gli1 in BxPC-3 cells, we detected the expressions of Gli1-targeted genes, such as Ptc1, CCND1 and BCL-2, compared with resveratrol experimental group. We further used the luciferase reporter assay to explore the correlation between resveratrol and Gli1. Results: Resveratrol inhibited the growth of pancreatic cancer cells in a dose- and time-dependent manner. Compared with control group, the cells in the G0/G1 phase and the apoptosis rate were significantly increased. Low concentration of resveratrol decreased the expression of the Hedgehog pathway members including Gli1, Ptc1 and Smo. The expression of downstream target genes of the Hedgehog pathway such as Gli1, Ptc1, CCND1 and BCL-2 were significantly decreased after 12.5 µM resveratrol treatment, which demonstrated a similar change of gene expression when Gli1 was knocked down by the RNAi technique in PANC-1 cells. Resveratrol also downregulated the expression of Gli1, Ptc1, CCND1 and BCL-2 in Gli1-overexpressed BxPC-3 cells. Results of the luciferase assay showed that resveratrol did not act on the Gli1 promoter directly. Conclusion: Resveratrol can inhibit pancreatic cancer cell survival and its mechanisms might be partly via the Hedgehog signaling pathway.