Tobacco smoking is an independent risk factor for the initiation of pancreatic cancer (PC). Hyper-methylation of tumor suppressor genes has been demonstrated to be associated with smoking. This study aimed to find the relationship between nicotine exposure and hypermethylation of tumor suppressor genes in normal pancreatic epithelial cells. Human pancreatic epithelial cells ware cultured exposing to nicotine and the methylation status of tumor suppressor genes were detected. Proenkephalin (PENK) was chosen as the target gene and methylation level of PENK promoter region was measured. Expression of DNA methyltransferase (DNMT), nicotine acetylcholine receptor (alpha 7nAChR) and signaling pathway downstream were analyzed. Nicotine induces overexpression of DNMT3A and 3B, and methylated-inactivation of PENK gene in normal pancreatic epithelial cells. An activation of alpha 7nAChR and MAPK signaling pathway has been detected in the nicotine-treated group. Demethylated drug, antagonist of alpha 7nAChR and inhibitor of p38 MAPK is verified to attenuate the overexpression of DNMTs stimulated by nicotine as well as inhibit aberrant hypermethylation-related silence of PENK gene. Nicotine stimulation can induce aberrant hypermethylation of tumor suppressor genes by alpha 7nAChR and MAPK signaling pathway-mediated up-regulation of DNMTs in pancreatic epithelial cells, thus we can provide epigenetic evidence of the mechanisms by which smoking causes pancreatic cancer and find new therapeutic target. (C) 2018 Elsevier Inc. All rights reserved.