Previous studies have demonstrated that microRNA (miRNA) expression is altered in human cancer. However, the molecular mechanism underlying these changes in miRNA expression remains unclear. In this study, we investigated the epigenetic modification of miR-124 genes and the potential function of miR-124 in pancreatic cancer. Using pyrosequencing analysis, we found that miR-124 genes (including miR-124-1, miR-124-2 and miR-124-3) are highly methylated in pancreatic cancer tissues compared with in non-cancerous tissues. Hypermethylation mediated the silencing of miR-124, which was a frequent event in pancreatic duct adenocarcinoma (PDAC). Furthermore, miR-124 downregulation was significantly associated with worse survival of PDAC patients. Functional studies showed that miR-124 inhibited cell proliferation, invasion and metastasis. Furthermore, we characterized Rac1 as a direct target of miR-124, and miR-124 interacted with the 3'-untranslated region of Rac1, which we showed to be a putative tumor promoter in pancreatic cancer. Thus, the miR-124-mediated downregulation of Rac1 led to the inactivation of the MKK4-JNK-c-Jun pathway. Therefore, our study demonstrates that miR-124 is a tumor suppressor miRNA that is epigenetically silenced in pancreatic cancer. Our findings suggest a previously unidentified molecular mechanism involved in the progression and metastasis of pancreatic cancer.