Background: Epigenetic regulation plays an important role in the initiation, promotion and progression of cancers. The alterations of DNA methylation and histone modification are frequently found in pancreatic cancer. However, their contribution to gemcitabine resistance is relatively unclear. Method: We investigated the expression of epigenetic regulators in PANC 1 and gemcitabine-resistant PANC-1 human pancreatic cancer cells by PCR-array. The role of the epigenetic regulators in the control of gemcitabine resistance was addressed by genetic manipulation or chemical inhibitors, and various functional assays were performed to elucidate the underlying mechanism. Results: We have identified EHMT2, a histone H3 lysine 9 (H3K9) methyltransferase, as a potential gene involved in gemcitabine resistance. Overexpression of this gene increased drug resistance in parental cells while knockdown sensitized resistant cells to gemcitabine. In addition, EHMT2 contributed to the stemness properties of pancreatic cancer cells. Finally, our animal study clearly demonstrated that the EHMT2 inhibitor could override the gemcitabine resistance in vivo. Conclusion: EHMT2 is upregulated in gemcitabine-resistant pancreatic cancer cells and is a potential target to overcome the resistance for cancer therapy.