Pancreatic cancer has a poor prognosis because of its high invasiveness and recurrence, and these properties closely link to the phenomenon of epithelial-mesenchymal transition (EMT). Recently, it has been reported that Sox4 is indispensable for EMT in vitro and in vivo and regulates various master regulators of EMT including Zeb, Twist and Snail. Moreover, Sox4 induces the transcription of Ezh2 which is the histone methyltransferase, and reprograms the cancer epigenome to promote EMT and metastasis. Therefore, the present study evaluated the importance of Sox4, Ezh2 and miR-335, which regulate Sox4 expression epigenetically, in clinical samples with pancreatic cancer. This retrospective analysis included data from 36 consecutive patients who underwent complete surgical resection for pancreatic cancer and did not undergo any preoperative therapies. We assessed the clinical significance of Sox4/Ezh2 axis and miR-335 expression, using immunohistochemistry and qRT-PCR with laser captured microdissection (LCM). The Sox4 positive patients had significantly worse prognosis as for disease-free survival (DFS) (P=0.0154) and the Ezh2-positive patients had significantly worse prognosis as for overall survival (OS) (P=0.0347). The miR-335 expression was inversely correlated with Sox4 expression in the identical clinical specimens, but it was not related to the prognosis. Sox4/Ezh2 axis was closely associated with the prognosis in pancreatic cancer patients.