Background Pancreatic cancer is obstinate and resistant to gemcitabine, a standard chemotherapeutic agent for the disease. We previously showed a therapeutic effect of glycogen synthase kinase-3 beta (GSK3 beta) inhibition against gastrointestinal cancer and glioblastoma. Here, we investigated the effect of GSK3 beta inhibition on pancreatic cancer cell sensitivity to gemcitabine and the underlying molecular mechanism. Methods Expression, phosphorylation, and activity of GSK3 beta in pancreatic cancer cells (PANC-1) were examined by Western immunoblotting and in vitro kinase assay. The combined effect of gemcitabine and a GSK3 beta inhibitor (AR-A014418) against PANC-1 cells was examined by isobologram and PANC-1 xenografts in mice. Changes in gene expression in PANC-1 cells following GSK3 beta inhibition were studied by cDNA microarray and reverse transcription (RT)-PCR. Results PANC-1 cells showed increased GSK3 beta expression, phosphorylation at tyrosine 216 (active form), and activity compared with non-neoplastic HEK293 cells. Administration of AR-A014418 at pharmacological doses attenuated proliferation of PANC-1 cells and xenografts, and significantly sensitized them to gemcitabine. Isobologram analysis determined that the combined effect was synergistic. DNA microarray analysis detected GSK3 beta inhibition-associated changes in gene expression in gemcitabine-treated PANC-1 cells. Among these changes, RT-PCR and Western blotting showed that expression of tumor protein 53-induced nuclear protein 1, a gene regulating cell death and DNA repair, was increased by gemcitabine treatment and substantially decreased by GSK3 beta inhibition. Conclusions The results indicate that GSK3 beta inhibition sensitizes pancreatic cancer cells to gemcitabine with altered expression of genes involved in DNA repair. This study provides insight into the molecular mechanism of gemcitabine resistance and thus a new strategy for pancreatic cancer chemotherapy.