Indiana University-Purdue University (IUPUI) / Most pancreatic cancer patients receiving gemcitabine chemotherapy eventually develop resistance to gemcitabine. To improve survival and prognosis of pancreatic cancer patients, better understanding the mechanisms of gemcitabine resistance and discovery of new therapeutic targets are required. In this study, I investigated the molecular mechanisms of acquired gemcitabine resistance using a stepwise gemcitabine-selected pancreatic cancer cell line in comparison to the parental cell line. I found that 14-3-3σ is up-regulated in the drug resistant cell line due to demethylation in its first exon, and the up-regulation of 14-3-3σ gene expression, in turn, contributes to gemcitabine resistance. Intriguingly, I found that demethylation of the 14-3-3σ gene in gemcitabine resistant cells is reversibly regulated by DNMT1 and UHRF1. Furthermore, I found that 14-3-3σ over-expression causes gemcitabine resistance by inhibiting gemcitabine-induced apoptosis and caspase-8 activation possibly via binding to YAP1. The finding of demethylation of the 14-3-3σ gene in gemcitabine resistant cells led to a hypothesis that other genes may also be changed epigenetically following gemcitabine selection. By RRBS (Reduced Representation Bisulfite Sequencing) analysis, 845 genes were found to have altered methylation. One of these genes, PDGFD, was further investigated and found to have reversible demethylation at its promoter region in the drug resistant cells and contribute to gemcitabine resistance possibly via autocrine activation of the STAT3 signaling pathway. Together, these findings not only provide evidence that 14-3-3σ and PDGFD over-expression contribute to acquired gemcitabine resistance and that reversible epigenetic changes may play an important role in acquired gemcitabine resistance, but also demonstrate that the molecular mechanisms of acquired gemcitabine resistance in pancreatic cancer cells are complex and multifaceted.