The dysregulation of microRNA (miRNA) expression has been highlighted in a variety of human malignant conditions with reports implicating a critical role in the process of tumor growth. The role of miR-539 in pancreatic cancer (PC) is yet to be fully elucidated, hence the aim of the current study was to investigate the effect of miR-539 expression in relation to a cohort of 52 PC specimens. The application of a real-time quantitative polymerase chain reaction (qRT-PCR) revealed a significantly down-regulated miR-539 level, which was accompanied by an increased TWIST1 expression in PC when compared with the controls. The in vitro experiment results demonstrated that the endogenic mimic of miR-539 significantly suppressed the growth of the xenograft tumors in PANC-1 cells, when compared to the delivery of the control miRNA and blank control. Meanwhile, the key epithelial-mesenchymal transition (EMT) inducer, TWIST1 was verified as a direct target gene of miR-539 through the application of a luciferase reporter assay. In conclusion, the results of the current study present evidence emphasizing the significance of the interactions between miR-539 and TWIST1 in the development of and progression of PC, highlighting its potential as a therapeutic target in the treatment of PC patients.